No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Reserpine modulates neurotransmitter release to extend lifespan and alleviate age-dependent Aβ proteotoxicity in Caenorhabditis elegans
Abstract
Aging is a debilitating process often associated with chronic diseases such as diabetes, cardiovascular and neurodegenerative diseases like Alzheimer's disease (AD). AD occurs at a very high incidence posing a huge burden to the society. Model organisms such as C. elegans become essential to understand aging or lifespan extension - the etiology, molecular mechanism and identification of new drugs against age associated diseases. The AD model, manifesting Aβ proteotoxicity, in C. elegans is well established and has provided valuable insights. Earlier, we have reported that Reserpine, an FDA-approved antihypertensive drug, increases C. elegans lifespan with a high quality of life and ameliorates Aβ toxicity in C. elegans. But reserpine does not seem to act through the known lifespan extension pathways or inhibition of its known target, vesicular monoamine transporter, VMAT. Reserpine's mode of action and the pathways it activates are not known. Here, we have evaluated the presynaptic neurotransmitter(s) release pathway and identified acetylcholine (ACh) as the crucial player for reserpine's action. The corroborating evidences are: i) lack of lifespan extension in the ACh loss of function (hypomorphic) - synthesis (cha-1) and transport (unc-17) mutants; ii) mitigation of chronic aldicarb effect; iii) lifespan extension in dopamine (cat-2) and dopamine and serotonin (bas-1) biosynthetic mutants; iv) no rescue from exogenous serotonin induced paralysis in the AD model worms; upon reserpine treatment. Thus, modulation of acetylcholine is essential for reserpine's action.
... Compounds in M9 buffer at a volume of 200 ul were spread on four-cm OP50 E. coli lawn containing NGM plates. 19,29 The worms were transferred to these plates after half an hour. Dose response was carried out for all the compounds. ...
... The standard aldicarb 19,30,31 assay was carried out with slight modifications. Aldicarb (0.1 mM) plates were prepared ½ h before the assay by spreading on the OP50 containing NGM agar plate. ...
... Levamisole assays were carried out as per procedures. 19,20 NGM plates with 200μM levamisole were prepared half an hour before the assay. On day one, adult worms were used for assay. ...
Background
Alzheimer’s disease (AD), a prevalent neurodegenerative disease with progressive dementia and neurotransmission (NT)-dysfunction-related complications in older adults, is known to be caused by abnormal Amyloid-β (Aβ) peptide and associated amyloid plaques in the brain. Drugs to cure AD are not in sight. Two major excitatory neurotransmitters, glutamate (Glu) and acetylcholine (ACh), and their signaling systems are implicated in AD.
Objective
To determine the effect of various NT-altering compounds including fenobam, quisqualic acid, and dimethyl sulfoxide (DMSO) in the protection against Aβ toxicity. Further, to identify the potential mechanism through which the protection happens.
Methods
The well-known C. elegans AD model, CL4176, in which human Aβ expression is turned on upon a temperature shift to 25 °C that leads to paralysis, was screened for protection/delay in paralysis because of Αβ toxicity. While screening the compounds, dimethyl sulfoxide (DMSO), a universal solvent used to solubilize compounds, was identified to provide protection. Aldicarb and levamisole assays were performed to identify the contribution of ACh neurotransmission in Αβ toxicity protection by DMSO.
Results
One percent and two percent DMSO delayed paralysis by 48% and 90%, respectively. DMSO was dominant over one of the Glu-NT pathway-related compounds, Fenobam-Group I mGluR antagonist. But DMSO provided only 30% to 50% protection against Quisqualic acid, the Glu-agonist. DMSO (2%) delayed ACh-NT, both presynaptic acetylcholine esterase inhibitor (AchEi)-aldicarb and postsynaptic-iAChR-agonst-levamisole induced paralysis, by ∼70% in CL4176. DMSO seems to be altering Ca ²⁺ ion permeability essential for NT as EthyleneDiamine Tetra-Acetic acid (EDTA) and DMSO provided similar aldicarb resistance either combined or alone in wildtype worms. But postsynaptic Ca ²⁺ depletion by EDTA could reverse DMSO-induced levamisole hypersensitivity. Surprisingly, the absence of FOrkhead boXO (FOXO) transcription factor homolog, daf-16 (loss-of-function mutant), a critical transcription factor in the reduced IIS-mediated longevity in C. elegans, abolished DMSO-mediated Ald R .
Conclusion
DMSO and Fenobam protect against Aβ toxicity through modulation of NT.
... C. elegans is a very good model for preclinical drug discovery, especially neurodegenerative diseases like Alzheimer disease [6][7] for which the current treatment is inhibition of the excitatory neurotransmitter, acetylcholine, degrading enzyme, acetylcholine esterase. One of the very simple, yet powerful assay for identification of acetylcholine release modulation at the neuromuscular junction of C. elegans is the utilization of acetylcholine esterase (AChE) inhibitor, aldicarb [5,8,7]. ...
... C. elegans is a very good model for preclinical drug discovery, especially neurodegenerative diseases like Alzheimer disease [6][7] for which the current treatment is inhibition of the excitatory neurotransmitter, acetylcholine, degrading enzyme, acetylcholine esterase. One of the very simple, yet powerful assay for identification of acetylcholine release modulation at the neuromuscular junction of C. elegans is the utilization of acetylcholine esterase (AChE) inhibitor, aldicarb [5,8,7]. Because of the AChE inhibition, acetylcholine accumulates at the neuromuscular junction (NMJ) leading to continuous activation of the ionotropic ACh receptors in the muscle leading to paralysis. ...
... Differential fractionation with organic solvents like n-hexane, ethanol, chloroform and ethyl acetate is very common in medicinal chemistry, bioprospecting for active biomolecules in the mega cocktail of natural products (Fain et al., 2017). The false positive effects of processed organic solvents which acts as vehicle control in the aldicarb assays ( Fig. 1 and Fig. 2) of C.elegans (Saharia et al., 2012) is implicating the necessity for extreme caution in all drug screening bioassays, given the enormous cost and the time frame of more than a decade and a half for drug discovery. ...
Background:
Identification of novel drugs by bio-prospecting natural products like various parts of the plants, or other extracts and drug discovery requires differential fractionation with various organic solvents followed by their concentration through evaporation under nitrogen gas, which is a standard practice.
Purpose:
Determination of contribution of vehicle control of organic solvents (chloroform, ethanol, ethyl acetate and n-hexane) processed in the similar manner in the modulation of acetylcholine(ACh) neurotransmission in Caenorhabditis elegans, Aldicarb induced paralysis assay.
Methods:
The organic solvents concentrated as described in background was used to identify their contribution in ACh modulation through ACh esterase inhibitor, Aldicarb, treatment of C. elegans, which leads to time dependent paralysis of the worms.
Results:
The vehicle, organic solvents, control itself bestows modulation of acetylcholine release as Aldicarb resistance in C. elegans.
Conclusion:
Given the exorbitant cost and time taken for drug discovery, identification of efficacy of bioactive molecules fractionated through organic solvents and concentrated under nitrogen gas should have appropriate vehicle control as described above to avoid the rate of false positives. This is universally applicable whether the drug is chemically synthesized or purified from natural products.
... Moreover, CAT-1 can package other biogenic amines for release, including serotonin and octopamine, both of which are synthesized in the nematode (Avery and Horvitz, 1990;Duerr et al., 1999). Finally, reserpine has been reported to have actions on other targets in the worm beyond CAT-1 (Saharia et al., 2012;Reckziegel et al., 2016). ...
... A limit to the utility of reserpine is the role that CAT-1 plays in packaging serotonin and octopamine for release (Duerr et al., 1999). Additionally, the assumption that reserpine actions reflect solely an antagonism of CAT-1 can be questioned (Saharia et al., 2012;Reckziegel et al., 2016). Finally, the need for chronic incubations of animals with reserpine to affect DA depletion raises the possibility of compensatory changes in neuronal development that cloud interpretations of whether recovered mutations induce long-lasting compensatory changes that may or may not reflect a role in ongoing DA signaling. ...
... In our prior forward genetic studies, we used relatively high concentrations (600 μM) of reserpine and grew animals from initial lysis to late L4s prior to Swip studies, which we speculated could induce developmental and behavioral changes that might lead to false conclusions as to mutation specificity as DA-related. This hypothesis is bolstered by findings that chronic reserpine incubations can impact worm survival and stress responses (Saharia et al. 2012(Saharia et al. , 2016Srivastava et al., 2008). Recently, Reckziegel et al. (2016) studied reserpine-induced toxicity in worms, revealing many developmental defects and behavioral modifications including changes in egg laying, defecation cycles and locomotion rate on food. ...
The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans, DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Loss of function mutations in genes that support DA biosynthesis, DA vesicular packaging and DA action at the extrasynaptic D2-type DA receptor DOP-3 suppress Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by βPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling.
... The endocrine axis of LE, mainly as reduced insulin/IGF-1 receptor, daf-2 signalling is well known and one of the best LE pathways in C. elegans and the mechanism of which is found to be consistent in yeast, fruit fly, mice and humans as evidenced in Centenarians (Kenyon 2010). But the contribution and mechanism of neurotransmitters, except for serotonin and partly acetylcholine (Srivastava et al. 2008;Saharia et al. 2012), in neither determining the lifespan nor the increase in longevity is known. Recently, International Space Research Mission has also reinforced the role of neuronal signalling, especially, acetylcholine, in increasing the lifespan in C. elegans (Honda et al. 2012). ...
... Recently, International Space Research Mission has also reinforced the role of neuronal signalling, especially, acetylcholine, in increasing the lifespan in C. elegans (Honda et al. 2012). Reserpine has also been shown to influence the lifespan of worms along similar lines by modulating acetylcholine release (Saharia et al. 2012). But the mechanism, especially, for reserpine mediated lifespan extension (RMLE) is not completely known. ...
... The loss of another major neurotransmitter, dopamine, as evidenced in the (loss of function) dopamine synthesis, tyrosine hydroxylase mutant, cat-2 (Sulston et al. 1975) did not affect RMLE (Saharia et al. 2012). But serotonin is partly required for RMLE (Srivastava et al. 2008) as observed in the serotonin synthesis-tryptophan hydroxylase, tph-1 (Sze et al. 2000) mutant. ...
Lifespan extension is an all systems encompassing event. Involvement of reduced insulin/IGF1 signalling is well worked out, first in the model organism Caenorhbaditis elegans followed by other systems including humans. But the role of neuronal component in lifespan extension is not well understood due to the refractory nature of neurons to small RNA interference (sRNAi) in C. elegans. Earlier, we have demonstrated that an antihypertensive drug, reserpine, extends lifespan through modulation of neurotransmitter release, especially, acetylcholine, in C. elegans. Intriguingly, the reserpine mediated lifespan extension (RMLE) does not happen through the known longevity pathways. Here, we report that the D2-type dopamine receptor (DOP-3), which acts through the inhibitory G-protein coupled (Gαi) pathway mediated signalling is partly required for RMLE. In the dop-3 loss of function mutant RMLE is shortened. DOP-3 acts through Gαo (goa-1). One of the downstream targets of G protein signalling is the transcription factor, jun-1. MRP-1, an ATP binding cassette transporter, belonging to the multidrug resistance protein family is one of the genes turned on by JUN-1. RMLE is shortened in dop-3→goa-1→jun1→mrp-1 loss of function mutants, elucidating the contribution of dop-3 signalling. The dop-3 receptor system is known to inhibit acetylcholine release. This suggests dopamine receptor, dop-3 could be contributing to the modulation of acetylcholine release by reserpine. ERI-1 is a 3′-5′ exoribonuclease, one of the negative regulators of sRNAi, whose loss of function makes neurons amenable to siRNA. In the absence of eri-1, RMLE is shortened. In the dop-3 loss-of-function background, lack of eri-1 completely abolishes RMLE. This suggests that dop-3 and eri-1 act in independent parallel pathways for RMLE and these two pathways are essential and sufficient for the longevity enhancement by reserpine in C. elegans.
... Various pharmaceutical, natural, supplementary therapies are being tried for AD [12,13] . Some of the natural compounds-alkaloid-reserpine [2] , components of red seaweed [14] , lycopene [15] , 18-alpha glycyrrhetinic acid proteasome activator [16] , traditional Chinese medicines such as Shengmai [17] and Liuwei Dihuang [18] -were reported to alleviate Aβ toxicity in the C. elegans model. ...
... Of these, earlier studies have reported that reserpine, an FDA approved anti-hypertensive and anti-psychotic drug, which also happens to be an Ayurvedic drug called 'sarpagandha' from the roots of Rauwolfia serpentina , alleviates amyloid toxicity in the above described model in C. elegans [1,2] . ...
... Reserpine reduces the plaque forming Aβ42 significantly in the Tg2576 Tg mouse model of AD [19] and improves the working memory [20] in the 5XFAD AD Tg mouse model [8] . Moreover, earlier studies have shown that reserpine modulates acetylcholine release in C. elegans [2] . However, the mechanism, genes, and pathways are not known. ...
Background:
With an incidence of 1 in 85 persons above the age of 60 years succumbing to the disease, Alzheimer's disease (AD), has been predicted to create havoc globally. In spite of enormous efforts and exhaustive research, no cure is in sight. Hence, it is critical to unravel the mechanism of AD development/protection and identification of a cure soon.
Purpose:
This study is aimed at investigating the mechanism of reserpine action, which alleviates the toxicity of amyloid beta (Aβ) (AD-causing peptide) in Caenorhabditis elegans [1, 2].
Methods:
Determination of alleviation of Aβ toxicity with reserpine manifested as reduction in progressive paralysis, in the background of GFP reporter driven by the promoter of the FMRFamide neuropeptide, FLP-11 (AD; Pflp-11::GFP) and acetylcholine contribution through aldicarb (which inhibits acetylcholine esterase) treatment.
Results:
The most significant protection against Aβ toxicity was obtained in the background of Pflp-11::GFP. This protection had 2 components. The promoter of FLP-11 with the reporter GFP, Pflp-11::GFP, per se gave significant protection. Further reserpine treatment provided additional alleviation. Together they could almost eliminate Aβ toxicity. These 2 components of Aβ toxicity alleviation are dependent on acetylcholine levels, as an increase in acetylcholine by aldicarb treatment reduces the protective effect.
Conclusion:
A unique way to alleviate Aβ toxicity is reserpine treatment in combination with Pflp-11::GFP. Reserpine should be evaluated as a potential drug in a pilot study in AD patients. Furthermore, identification of the mechanism of Pflp-11::GFP-mediated reduction in Aβ toxicity is a potential pathway to develop therapeutics for AD.
... This normally transports free norepinephrine, serotonin and dopamine from the cytoplasm of the presynaptic nerve terminal into storage vesicles for subsequent release into the synaptic cleft. Unprotected neurotransmitters are metabolized by MAO in the cytoplasms and consequently never reach the synapse [36]. Reserpine administration to mice were diminised the brain norepinephrine, dopamine and serotonin levels. ...
... This normally transports free norepinephrine, serotonin and dopamine from the cytoplasm of the presynaptic nerve terminal into storage vesicles for subsequent release into the synaptic cleft. Unprotected neurotransmitters are MAO in the cytoplasms and consequently never reach the synapse [36]. Reserpine administration to mice were diminised the brain norepinephrine, dopamine and serotonin levels. ...
... Resistance of the CL2006 strain to ACh agonists is due to production and deposition of both Aβ oligomers and fibrils 59 . Exposure to aldicarb (Fig 2A) results in paralysis within 180 min, which, as expected, occurs more rapidly under dld-1 gene suppression (within 120 min, p=0.0001). ...
... Accumulation of Aβ oligomers is thought to be a major culprit in AD progression 73,74 , whereas monomers actually help to maintain glucose homeostasis and are not toxic 75,76 . Our findings suggest that dld-1 suppression reduces Aβ oligomerization thus resulting in reduced paralysis, better movement rates, and improved behavioral phenotypes as observed previously 43,59,77,78 . Both mutation and RNAi-mediated suppression of the dld-1 gene result in phosphine resistance and an extended lifespan 38,39,78 , as well as inhibition of Aβ oligomerization and protection against Aβ-mediated toxicity as we have shown here. ...
A decrease in energy metabolism is associated with Alzheimer’s disease (AD), but it is not known whether the observed decrease exacerbates or protects against the disease. The importance of energy metabolism in AD is reinforced by the observation that variants of dihydrolipoamide dehydrogenase (DLD), which is a subunit of both complexes, is genetically linked to late-onset AD. To determine whether DLD is a suitable therapeutic target, we suppressed the dld-1 gene in C. elegans that express human Aβ peptide in either muscles or neurons. Suppression of the dld-1 gene resulted in significant restoration of vitality and function that had been degraded by Aβ pathology. This included protection of neurons and muscles cells. The observed decrease in proteotoxicity was associated with a decrease in the formation of toxic oligomers rather than a decrease in the abundance of the Aβ peptide. The mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which like dld-1 gene expression inhibits ATP synthesis, had no significant effect on Aβ toxicity. Proteomics data analysis revealed that beneficial effects after dld-1 suppression could be due to change in energy metabolism and activation of the pathways associated with proteasomal degradation, improved cell signaling and longevity. Thus, some feature unique to dld-1 gene suppression is responsible for the therapeutic benefit. By direct genetic intervention, we have shown that acute inhibition of dld-1 gene function may be therapeutically beneficial. This result supports the hypothesis that lowering energy metabolism protects against Aβ pathogenicity and that DLD warrants further investigation as a therapeutic target.
... Aldicarb is an acetylcholine esterase inhibitor, which causes increased accumulation of acetylcholine esterase (AChE) at the synapse that leads to paralysis in the worms (Rand and Russell, 1984;Saharia et al., 2012). The presence of AChE is also associated with Aβ plaques formation, which is one of the common features in the AD-associated pathologies (Saharia et al., 2012). ...
... Aldicarb is an acetylcholine esterase inhibitor, which causes increased accumulation of acetylcholine esterase (AChE) at the synapse that leads to paralysis in the worms (Rand and Russell, 1984;Saharia et al., 2012). The presence of AChE is also associated with Aβ plaques formation, which is one of the common features in the AD-associated pathologies (Saharia et al., 2012). Pharmacological interventions have shown that phytomolecules supplementation could delay the AChE-induced paralysis phenotype in worms Smita et al., 2017). ...
Betula utilis (BU), an important medicinal plant that grows in high altitudes of the Himalayan region, has been utilized traditionally due to it's antibacterial, hepatoprotective, and anti-tumor properties. Here, we demonstrated the longevity and amyloid-β toxicity attenuating activity of B. utilis ethanolic extract (BUE) in Caenorhabditis elegans. Lifespan of the worms was observed under both the standard laboratory and stress (oxidative and thermal) conditions. Effect of BUE was also observed on the attenuation of age-dependent physiological parameters. Further, gene-specific mutants and green fluorescent protein (GFP)-tagged strains were used to investigate the molecular mechanism underlying the beneficial effects mediated by BUE supplementation. Our results showed that BUE (50 μg/ml) extended the mean lifespan of C. elegans by 35.99% and increased its survival under stress conditions. The BUE also reduced the levels of intracellular reactive oxygen species (ROS) by 22.47%. A delayed amyloid-β induced paralyses was observed in CL4176 transgenic worms. Interestingly, the BUE supplementation was also able to reduce the α-synuclein aggregation in NL5901 transgenic strain. Gene-specific mutant studies suggested that the BUE-mediated lifespan extension was dependent on daf-16, hsf-1, and skn-1 but not on sir-2.1 gene. Furthermore, transgenic reporter gene expression assay showed that BUE treatment enhanced the expression of stress-protective genes such as sod-3 and gst-4. Present findings suggested that ROS scavenging activity, together with multiple longevity mechanisms, were involved in BUE-mediated lifespan extension. Thus, BUE might have potential to increase the lifespan and to attenuate neuro-related disease progression.
... Deposition of Aβ in the AD brain is associated with disease progression, whereas deposition in human muscles in old age results in inclusion-body myositis that impairs proper muscle functioning [38,39]. In C. elegans strain CL4176, expression of Aβ in muscle is temperature inducible, which results in time-dependent deposition of Aβ as well as a timedependent paralysis phenotype [37,38,40]. In this study, we found that inclusion of metformin in the growth medium delayed the onset of paralysis in a dose-dependent manner (Fig. 1). ...
... Aβ expression in muscle is known to impair acetylcholine neurotransmission in C. elegans [40]. As such, the ability of metformin treatment to delay Aβ-mediated paralysis may be the result of improved acetylcholine neurotransmission, an essential element of muscle contraction [41]. ...
... Moreover, reserpine has always been considered to be an effective antihypertensive, even for older patients [29]. In a situation where the world is aging, life expectancy is increasing, majority of the patients on reserpine treatment are between 50 and 70 years old in the poor, rural Related to increased health span, it has been identified that reserpine could provide high quality lifespan extension in C. elegans [30,31]. In addition, reserpine was able to provide protection against dementia -Alzheimer disease (a geriatric disease affecting many old people, known to be a huge social and financial burden on society) causing, Aβ-induced toxicity manifested as progressive paralysis, in the C. elegans model [31][32][33]. ...
... In a situation where the world is aging, life expectancy is increasing, majority of the patients on reserpine treatment are between 50 and 70 years old in the poor, rural Related to increased health span, it has been identified that reserpine could provide high quality lifespan extension in C. elegans [30,31]. In addition, reserpine was able to provide protection against dementia -Alzheimer disease (a geriatric disease affecting many old people, known to be a huge social and financial burden on society) causing, Aβ-induced toxicity manifested as progressive paralysis, in the C. elegans model [31][32][33]. Similarly, in the AD mouse model, Tg2576, expressing human mutant APP, which causes Aβ aggregates formation and cognitive deficits at ~11 months of age, reserpine reduces the Aβ levels [34] and enhances working memory [35] in the 5XFAD AD model [36]. ...
... The adult wildtype and CL2006 worms were washed off with M9 and placed on aldicarb plate and scored paralysis for 6 h at the interval of every 30 min. [27,28] The worms which did not response to repeated prodding considered as paralyzed. ...
... Previous studies have reported that bioactive molecules with longevity promoting and stress modulating properties also possess neuroprotective effect and reduce Aβ toxicity in C. elegans. [28,35,36] We have recently reported anti-aging and stress modulating potential of flavonoid DMF in wildtype C. elegans. [10] Therefore, the present article focused on the protective effects of DMF against AD by targeting Aβ toxicity and oxidative stress using transgenic C. elegans. ...
Background: The various herbal remedies have been used in Ayurveda which symbolizes traditional medicine system since ancient times. The increasing popularity of herbal medicines has prompted us toward the development of natural therapeutics for preventing neurodegenerative disease such as Alzheimer's disease (AD) in living organisms. This study focused on a flavonoid compound 5,7-dihydroxy-4-methoxyflavone (DMF) also known as acacetin which is a major constituent of Premna odorata (L.) plant. This bioactive flavonoid exhibits several medicinal properties such as antimicrobial, anti-inflammatory, antioxidant, and anti-carcinogenic. Objective: The aim is to determine the protective effects of DMF against amyloid beta (Aβ)-induced toxicity and oxidative stress in transgenic Caenorhabditis elegans model of AD. Materials and Methods: The therapeutic potential of DMF treatments (5, 25, and 50 μM) was investigated to counteract Aβ paralysis and oxidative stress through paralysis assay, reactive oxygen species (ROS) detection, protein carbonylation, aldicarb assay, and mRNA quantification using transgenic C. elegans model of AD. Results: The present study reports that DMF effectively delayed Aβ-induced paralysis, attenuated ROS level reduced protein carbonylation and conferred aldicarb resistance. In addition, DMF was also found to up-regulate the expression of stress modulating (sod-1, sod-2, sod-3, ctl-1, hsp-16.2, and gst-4), acetylcholine transporter(unc-17), regulator of nicotinic acetylcholine receptor (unc-50), and choline acetyltransferase (cha-1) related genes. Conclusion: These findings suggest DMF may provide protection against Aβ toxicity and oxidative stress due to its antioxidant activity. Therefore, the bioactive flavonoid DMF may provide invaluable medicinal and health benefits which can delay the onset of age-related diseases.
... Deposition of Aβ in the AD brain is associated with disease progression, whereas deposition in human muscles in old age results in inclusion-body myositis that impairs proper muscle functioning [38,39]. In C. elegans strain CL4176, expression of Aβ in muscle is temperature inducible, which results in time-dependent deposition of Aβ as well as a timedependent paralysis phenotype [37,38,40]. In this study, we found that inclusion of metformin in the growth medium delayed the onset of paralysis in a dose-dependent manner (Fig. 1). ...
... Aβ expression in muscle is known to impair acetylcholine neurotransmission in C. elegans [40]. As such, the ability of metformin treatment to delay Aβ-mediated paralysis may be the result of improved acetylcholine neurotransmission, an essential element of muscle contraction [41]. ...
The metabolic disease, type 2 diabetes mellitus
(T2DM), is a major risk factor for Alzheimer’s disease (AD).
This suggests that drugs such as metformin that are used to treat
T2DM may also be therapeutic toward AD and indicates an
interaction between AD and energy metabolism. In this study,
we have investigated the effects of metformin and another
T2DM drug, 5-aminoimidazole-4-carboxamide ribonucleotide
(AICAR) in C. elegans expressing human Aβ42. We found that
Aβ expressed in muscle inhibited levamisole sensitive nicotinic
acetylcholine receptors and that metformin delayed Aβ-
linked paralysis and improved acetylcholine neurotransmission
in these animals. Metformin also moderated the effect of neuronal
expression of Aβ: decreasing hypersensitivity to serotonin,
restoring normal chemotaxis, and improving fecundity.
Metformin was unable to overcome the small effect of neuronal
Aβ on egg viability. The protective effects of metformin were
associated with a decrease in the amount of toxic, oligomeric
Aβ. AICAR has a similar protective effect against Aβ toxicity.
This work supports the notion that anti-diabetes drugs and metabolic
modulators may be effective against AD and that the
worm model can be used to identify the specific interactions
between Aβ and cellular proteins.
... In addition, an interesting review by Morcos and Hutter (2009) details the role of C. elegans in the furtherance of our understanding of the association between AD and DM, especially as it relates to the role of advanced glycation end products (AGEs) and oxidative stress in the progression of these diseases. Moreover, using this same nematode model, Saharia et al. (2012) recently demonstrated the beneficial effects of the antihypertensive drug riserpine in extending lifespan and reducing Aβ toxicity via a mechanism that involves modulation of acetylcholine action. In the same vein, Kumar et al. (2015) have reported the mechanisms via which silymarin extends lifespan and ameliorates proteotoxicity in the C. elegans model of Alzheimer's dementia to include modulation of oxidative stressa key player in ageing. ...
... Moreover, when treated with the antidiabetic drug metformin, increased lifespan was reported in a C. elegans model of with type 2 diabetes mellitus (Onken and Driscoll 2010). The use of C. elegans also lead to the understanding of the acetylcholine mechanism of action of reserpine, a drug used in the management of AD (Saharia et al. 2012). Therefore, as our use of nematode models of chronic diseases evolves, so will our understanding of the pathobiology of these distinct yet related pathologic entities. ...
7
As the burden of Alzheimer’s dementia rises, so does our understanding of the cellular and molecular basis of this neurodegenerative disease. Some of the recent advances in the aetiopathogenesis of neurodegeneration include the finding that insulin receptor signalling is key to neurogenesis and synaptogenesis in the brain, especially in areas related to memory formation and storage, including the hippocampus. This suggests an association between impaired insulin receptor signalling and neurodegenerative events. To decipher this association, several animal models are being employed. Such models include transgenic and non-transgenic animals that range from invertebrates (Drosophila melanogaster and Caenorhabditis elegans), to vertebrates (mouse, rats and primates). The current review is an account of such models and how they have contributed to our understanding of the relationship between type 2 diabetes mellitus, ageing and dementia.
... 4 So far, neither an innocuous longevity supplement nor a therapy to translate this longevity in humans is yet known. Recently, we reported an antihypertensive drug, reserpine, derived from the plant Rauwolfia serpentina, which is able to extend C. elegans lifespan 11,12 and alleviate Amyloid β toxicity in C. Elegans. 12,13 Consumption of red wine is suspected to delay aging. ...
... Recently, we reported an antihypertensive drug, reserpine, derived from the plant Rauwolfia serpentina, which is able to extend C. elegans lifespan 11,12 and alleviate Amyloid β toxicity in C. Elegans. 12,13 Consumption of red wine is suspected to delay aging. Later, resveratrol present in red wine is shown to extend lifespan in C. elegans 14 and mice, 6 and is being used as a nutritional supplement. ...
Background:
In the ancient Indian herbal medicine system several ayurvedic preparations are claimed to have longevity enhancing effects. But, so far, no clear scientific evidence has been provided. One among them, is the roots of the plant, commonly known as Ashwagandha (Withania somnifera Dunal- WSD), which is supposed to have myriad of beneficial effects including long life.
Purpose:
Here, we evaluated both the root extract (RE) and its purified ingredients (PI-RE) with a similar composition as in RE obtained from the roots of WSD for lifespan extension in the well established model system, C. elegans. PI-RE could extend the lifespan of C. elegans.
Methods:
We used wild type C. elegans (N2) or RB918: acr-16 (ok789); andNL2099: rrf-3 (pk1426) mutant worms and analysed their lifespan assay in Ashwagandha extract spreaded on plates containing Bacterial Lawns.
Results:
Strangely, while there was no effect on the wild type worms, the mutant for the human nicotinic acetylcholine receptor, nAchR, α7 equivalent, acr-16, showed around ~20% lifespan extension when treated with PI-RE.
Conclusion:
Thus, we are able to show that one of the age old healthy longlife supplements, Ashwagandha does extend lifespan of C. elegans.
... Furthermore, the increase in the time between defecation cycles, related to the increase in AChE activity with the inhibition of the excitability of the cholinergic system, allows the reduction of the defecation cycle 20 . Naturally, in C. elegans mutant strains for neurodegenerative diseases, as well as in patients affected by neurodegenerative diseases, there is a harmful decrease in the levels of neurotransmitters and also in the activity of enzymes that act in the synapse process 33 . To reverse this effect, defining compounds capable of activating and stabilizing these enzymes at normal levels is an important process to reduce the progression of neurodegenerative diseases. ...
Substances from the Cannabis sativa species, especially cannabidiol (CBD) and Delta‑9‑tetrahydrocannabinol (Δ9‑THC), have attracted medical attention in recent years. The actions of these two main cannabinoids modulate the cholinergic nervous system (CholNS) involving development, synaptic plasticity, and response to endogenous and environmental damage, as a characteristic of many neurodegenerative diseases. The dynamics of these diseases are mediated by specific neurotransmitters, such as the GABAergic nervous system (GNS) and the CholNS. The nematode Caenorhabditis elegans is an important experimental model, which has different neurotransmitter systems that coordinate its behavior and has a transgene strain that encodes the human β‑amyloid 1–42 peptide in body wall muscle, one of the main proteins involved in Alzheimer´s disease. Therefore, the objective of this study was to evaluate the protective potential of terpenoids found in C. sativa in the GNS and CholNS of C. elegans. The effect of two C. sativa oils with variations in CBD and THC concentrations on acetylcholinesterase (AChE) activity, lipid peroxidation, and behavior of C. elegans was evaluated. C. sativa oils were efficient in increasing pharyngeal pumping rate and reducing defecation cycle, AChE activity, and ROS levels in N2 strains. In the muscle: Abeta1-42 strain, mainly when using CBD oil, worm movement, body bends, and pharyngeal pumping were increased, with
a reduced AChE activity. Consequently, greater investments in scientific research are needed, in addition to breaking the taboo on the use of the C. sativa plant as an alternative for medicinal use, especially in neurodegenerative diseases, which have already shown positive initial results.
... Acetylcholine agonists are commonly used to treat the symptoms of AD [53] . Expression of A β in C. elegans muscles is associated with impaired ACh neurotransmission [54] . Given that glucose protects against A β-mediated paralysis in C. elegans , we were interested to determine whether this improvement was due to improved ACh signaling. ...
Amyloid beta (Aβ) plaques formation and impaired neurotransmission and neuronal behaviors are primary hallmarks of Alzheimer's disease (AD) that are further associated with impaired glucose metabolism in elderly AD's patients. However, the exact role of glucose metabolism on disease progression has not been elucidated yet. In this study, the effect of glucose on Aβ- mediated toxicity, neurotransmission and neuronal behaviors has been investigated using a C. elegans model system expressing human Aβ. In addition to regular diet, worms expressing Aβ were supplemented with different concentrations of glucose and glycerol and 5mM 2-deoxyglucose to draw any conclusions. Addition of glucose to the growth medium delayed Aβ-associated paralysis, promoted abnormal body shapes and movement, unable to restore impaired acetylcholine neurotransmission, inhibited egg laying and hatching in pre-existing Aβ-mediated pathology. The harmful effects of glucose may associate with an increase in toxic Aβ oligomers and impaired neurotransmission. O-β-GlcNAcylation (O-GlcNAc), a well-known post-translational modification is directly associated with glucose metabolism and has been found to ameliorates the Aβ- toxicity. We reasoned that glucose addition might induce O-GlcNAc, thereby protect against Aβ. Contrary to our expectations, induced glucose levels were not protective. Increasing O-GlcNAc, either with Thiamet-G (TMG) or by suppressing the O-GlcNAcase (oga-1) gene does interfere with and therefore reduce Aβ- toxicity but not in the presence of high glucose. The effects of glucose cannot be effectively managed by manipulating O-GlcNAc in AD models of C. elegans. Our observations suggest that glucose enrichment is unlikely to be an appropriate therapy to minimize AD progression.
... Dr Jamuna R. Subramaniam's group reported that Reserpine, an FDA-approved antihypertensive drug, could modulate neurotransmitter release to extend lifespan and alleviate age-dependent Aβ proteotoxicity in C. elegans. They further identified acetylcholine as the crucial player in reserpine's action, since reserpine could not extend lifespan in C. elegans without cha-1 expression [11]. The literature has also shown some relief effect of GPC on senescence, transthyretin deposition, and osteoarthritis in aged mice [12]. ...
Metabolism and aging are closely connected. The choline derivative glycerophosphocholine (GPC), an important precursor of the neurotransmitter acetylcholine, plays important roles in brain and nervous system function. Although it has been reported to alleviate cognitive decline in aged mice, whether GPC could promote longevity and other fitness factors remains unclear. Here, we find endogenous GPC level declines in the plasma of ageing humans. In Caenorhabditis elegans (C. elegans), GPC extends lifespan and improves exercise capacity during aging. Likewise, GPC inhibits lipofuscin accumulation. We further show that GPC treatment has no adverse effect on nematodes’ reproductive abilities and body length. In addition to its benefits under normal conditions, GPC enhances the stress resistance of C. elegans. Mechanically, we find GPC significantly inhibits the reactive oxygen species (ROS) accumulation in worms. Our findings indicate the health benefits of GPC and its potential application in strategies to improve lifespan and healthspan.
... Thus, the elevated activity of AChE is correlated with the elevation in its substrate Ach level in the neuronal tissues which could reflect the increase in cholinergic activity. It was reported that reserpine is able to modulate the presynaptic acetylcholine release [35] or has a central cholinomimetic properties [36]. This finding may indicate that reserpine chronic administration in the present study resulted in depletion of monoamines in brain tissue and additionally augmented the level of Ach which resulted in displaying the depressive-like symptoms in the animals. ...
The present study aimed at investigating the antidepressant and antioxidant actions of near-infrared (NIR) laser at a wavelength of 830 nm and power of 100 mW which applied transcranially on an animal model of depression induced by repeated doses of reserpine (0.2 mg/kg). Thirty male Wistar adult rats were divided into three groups: rat model of depression; rat model of depression irradiated with laser for 14 days after induction of depression; and the control group that was given the drug vehicle and sham-exposed to the laser. Forced swimming test (FST) was used to verify the induction of animal model of depression and to screen the effect of antidepressant effect of low-level laser at the end of the experiment. Monoamine level, oxidative stress markers, and activities of acetylcholinesterase (AchE) and monoamine oxidase (MAO) were determined in the cortex and hippocampus of the rat brain. Reserpine resulted in depletion of monoamines and elevation in the oxidative stress markers and change in the enzymatic activities measured in both brain areas. Laser irradiation has an inhibitory action on the monoamine oxidase (MAO) in the cortex and hippocampus leading to elevation of the monoamine levels and attenuation of the oxidative stress in the studied areas. FST has emphasized the antidepressant effect of the utilized laser irradiation parameters on the behavioral level. The present findings provide evidence for the antidepressant and antioxidant actions of NIR low-power laser in the rat model of depression. Accordingly, low-laser irradiation may be presented as a potential candidate modality for depression treatment.
... A nAChR agonist, levamisole was used to generate paralysis and measure the effect of SW on ACh transmission in worms post synaptically (Saharia et al., 2012). In brief, the agesynchronized embryos were shifted to the control and treatment plates. ...
The ailments related to a malfunction in cholinergic functioning currently employ the use of inhibitors for acetylcholinesterase (AChE) and N-methyl-d-aspartate (NMDA) receptors. The present study was designed to elucidate the potential of swertiamarin (SW), a secoiridoidal glycoside isolated from Enicostemma littorale in curtailing the cholinergic dysfunction. Using Caenorhabditis elegans as a model, SW was found to enhance neurotransmission by modulating AChE and nicotinic acetylcholine receptor (nAChR) activity; being orchestrated through up-regulation of unc-17 and unc-50. SW exhibited AChE inhibition both in vivo and cell-free system. The in silico molecular docking of SW and human AChE (hAChE) displayed good binding energy of −6.02. Interestingly, the increase in aldicarb and levamisole sensitivity post SW treatment was curtailed to a significant level in daf-16 and skn-1 mutants. SW raised the level of the endogenous antioxidant enzymes through up-regulation of sod-3 and gst-4 that act downstream to DAF-16 and SKN-1, imparting protection against neurodegeneration. The outcome of our study displays SW as a potential natural molecule for the amelioration of cholinergic dysfunction. Moreover, the study also indicates that SW elicits antioxidant response via up-modulation of daf-16 possibly through unc-17 upregulation. Further research on SW pertaining to the underlying mechanism and potential is expected to significantly advance the current understanding and design of possible ameliorative or near ameliorative regimens for cholinergic dysfunction.
... Independently, in a C. elegans screen, reserpine was found to extend lifespan 19 , albeit, through modulation of acetylcholine release 20 , partially through the action of Goi coupled dopamine receptor, dop-3 21 . More importantly, reserpine could protect against the Alzheimer disease causing Amyloid beta toxicity induced paralysis in C. elegans 22 and improved working memory in the 5XFAD Alzheimer disease model mice 23,24 suggesting reserpine as a potential drug to provide cognitive protection. ...
Change in diet life style and increased life expectancy has led to the dramatic escalation in old age related complication like cognitive decline leading to dementia. Cardiovascular diseases (CVD) are huge risk factors for dementia, including Alzheimer disease (AD). Hypertension is very well known to cause cognitive impairment. Control of CVD could provide protection against dementia. Earlier in the mouse model of AD, reserpine, an antihypertensive and antipsychotic drug could elicit improvement in the working memory in AD model mice and enhance the same in normal mice. Hence, Cognitive protection in the patients on chronic antihypertensive drug which contains reserpine was evaluated. Cognition in a cohort (in the South Indian rural population) of hypertensive patients (majority age group 50 to 70 years) who have been chronically treated with a combinatorial drug(adelphane/adelphane esidrexsodl by Novaritis Switzerland) consisting of reserpine and hydrazine for years was compared with controls without hypertension. The cohorts were age sex socio-economic education background matched. Cognition was scored using the Tamil version of: Addenbrooke Cognitive Examination-III (ACEIII (Tamil)) and Montreal Cognitive Assessment (Tamil) scales. The composite ACEIII (Tamil) score of control and treated groups were 53.6 and 53.2 respectively. MoCA(tamil) scores (Control :15.1 and Treated: 14.7) did not show much alteration. Further the mean scores of the control and treated groups individual components of cognition in ACE, namely Attention Memory Fluency Language and visuospatial cognitive skills also did not reveal significant difference. Thus controlling blood pressure or hypertension with chronic antihypertensive medication like adelphane/adelphane esirdex (reserpine containing drugs) has retained normal cognition in both genders.
... Bacillus subtilis alleviated the paralysis phenotype of transgenic C. elegans expressing the human Aβ peptide in muscle Caenorhabditis elegans offers a valuable platform for investigating the cellular and molecular mechanisms of AD [22,23]. The A is believed to be the major cause of AD pathogenesis, and its expression in transgenic C. elegans strains produces several pathological features important to better understand AD pathology [24,26,37,38]. Two of the transgenic AD C. elegans strains are CL2120 and GMC101, which express human A peptides of different sizes and toxicities, namely A 3-42 and A 1-42 , respectively [24,26]. ...
Multiple causes, apart from genetic inheritance, predispose to the production and aggregation of amyloid-β (Aβ) peptide and Alzheimer's disease (AD) development in the older population. There is currently no therapy or medicine to prevent or delay AD progression. One novel strategy against AD might involve the use of psychobiotics, probiotic gut bacteria with specific mental health benefits. Here, we report the neuronal and behavioral protective effects of the probiotic bacterium Bacillus subtilis in a Caenorhabditis elegans AD model. Aging and neuronal deterioration constitute important risk factors for AD development, and we showed that B. subtilis significantly delayed both detrimental processes in the wild-type C. elegans strain N2 compared with N2 worms colonized by the non-probiotic Escherichia coli OP50 strain. Importantly, B. subtilis alleviated the AD-related paralysis phenotype of the transgenic C. elegans strains CL2120 and GMC101 that express, in body wall muscle cells, the toxic peptides Aβ3-42 and Aβ1-42, respectively. B. subtilis-colonized CL2355 worms were protected from the behavioral deficits (e.g., poor chemotactic response and decreased body bends) produced by pan-neuronal Aβ1-42 expression. Notably, B. subtilis restored the lifespan level of C. elegans strains that express Aβ to values similar to the life expectancy of the wild-type strain N2 fed on E. coli OP50 cells. The B. subtilis proficiencies in quorum-sensing peptide (i.e., the Competence Sporulation Factor, CSF) synthesis and gut-associated biofilm formation (related to the anti-aging effect of the probiotic) play a crucial role in the anti-AD effects of B. subtilis. These novel results are discussed in the context of how B. subtilis might exert its beneficial effects from the gut to the brain of people with or at risk of developing AD.
... Two small reports additionally showed that Reserpine, a pleiotropic indole alkaloid drug that has been used both as an antipsychotic and antihypertensive agent, prevents Aβ toxicity and increases lifespan in a C. elegans AD model [117,118]. Its numerous side-effects probably dissuade further research in AD. ...
INTRODUCTION
Midlife hypertension has been consistently linked with increased risk of cognitive decline and Alzheimer’s disease (AD). Observational studies and randomized trials show that the use of antihypertensive therapy is associated with a lesser incidence or prevalence of cognitive impairment and dementia. However, whether anti-hypertensive agents specifically target the pathological process of AD remains elusive.
AREAS COVERED
This review of literature provides an update on the clinical and preclinical arguments supporting anti-AD properties of antihypertensive drugs. The authors focused on validated all classes of antihypertensive treatments such as angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), calcium channel blockers (CCB), β-blockers, diuretics, neprilysin inhibitors, and other agents. Three main mechanisms can be advocated: action on the concurrent vascular pathology, action on the vascular component of Alzheimer’s pathophysiology, and action on non-vascular targets.
EXPERT OPINION
In 2019, while there is no doubt that hypertension should be treated in primary prevention of vascular disease and in secondary prevention of stroke and mixed dementia, the place of antihypertensive agents in the secondary prevention of “pure” AD remains an outstanding question.
... Reserpine (Figure 9, a natural compound isolated from the roots of the plant Rauwolfia serpentina) is an FDA-approved antihypertensive drug. [65] It increases the lifespan and ameliorates the proteotoxic effect of Ab in C. elegans, as reported by Srivastava et al. [66] In a later report, Saharia et al. study the mechanism by which reserpine brings about lifespan extension or decreases the proteo-toxic effect of Ab. [67] They hypothesize that this compound brings about reserpine-mediated lifespan extension (RMLE) and alleviates Ab toxicity through either the muscarinic acetylcholine receptor(s) or ionotropic ACh receptors and/or other pathways. They report that modulation of acetylcholine levels can help in RMLE and can decrease Ab toxicity. ...
Alzheimer's disease (AD) is a progressive neurodegenerative disease with no clinically accepted treatment to cure or halt its progression. The Food and Drug Administration (FDA) approved drugs (rivastigmine, donepezil, galantamine and memantine) at best provide marginal benefits thus emphasize the urgent need to explore other molecular entities as future drug candidates for AD. Looking at the wide pharmaceutical applications of heterocyclic compounds and particularly those containing benzofuran and indole ring system; these molecular frameworks draw special attention of the medicinal chemists for further evaluation in numerous diseases. This article focuses on the history and recent advances of benzofuran and indole based compounds as inhibitors of butyrylcholinesterase (BuChE), acetylcholinesterase (AChE), γ‐secretase, β‐secretase, tau misfolding, and β‐amyloid (Aβ) aggregation.
... Because the presence of Aβ blocks the release of acetylcholine (Ach), worms that express Aβ are protected against the acute paralysis that is caused by hyperexcitation of the cholinergic system. Thus, acute paralysis caused by two cholinergic agonists, aldicarb, an ACh esterase inhibitor (pre-synaptic effect) and levamisole, a receptor agonist (post-synaptic effect) is delayed in strains that express Aβ (Diomede et al., 2014;Oh and Kim, 2017;Saharia et al., 2012;Xin et al., 2013). To check whether MICA restores normal ACh neurotransmission (i.e. ...
Alzheimer's disease (AD) is an age-related disease characterized by loss of memory and disrupted thinking that is associated with altered energy metabolism. Variants of an important enzyme of energy metabolism, dihydrolipoamide dehydrogenase (dld), have been genetically linked to late-onset AD. Moreover, reduced activity of DLD-containing enzyme complexes is associated with AD progression. To understand how energy metabolism influences AD progression, we exposed C. elegans expressing human Aβ peptide to the chemical inhibitor of DLD, 2-methoxyindole-5-carboxylic acid (MICA). Expression of human Aβ in C. elegans causes a variety of pathologies that can be used to monitor the efficacy of treatments against proteotixicity. We found that MICA alleviated the Aβ-induced paralysis and improved cholinergic neurotransmission in C. elegans that express Aβ in muscle cells. MICA also reduced both hypersensitivity to serotonin and perturbation of chemotaxis associated with neuronal expression of human Aβ. Furthermore, low doses of MICA helped to alleviate an Aβ-mediated decrease in fecundity. Protection against AD pathogenesis by MICA in the C. elegans model was associated with a decrease in Aβ oligomerization that could be reversed by the calcium ionophore, A23187. MICA also caused a decrease in oxidative stress, which could also contribute to the protective effect of MICA against Aβ toxicity.
... As non communicable diseases are in the rise due to aging, strategies to increase healthspan is rigorously evaluated using all possible systems and organisms. Generally, a gene or its absence or specific chemicals like Re- serpine [17] [18], Aswagandha [11], Rapamycin [19] and Resveratrol [20] can increase lifespan in various organisms like yeast, C. elegans, drosophila and mice. In addition, they induce stress tolerance. ...
Aging is a decelerating unidirectional process of life. Shortening of telomeric DNA, the (TTAGGG)n
hexanucleotide repeats, which form the caps at the chromosome ends, is implicated to determine
the aging process, and more importantly the healthy lifespan itself. Telomerase, a ribonucleoprotein
having reverse transcriptase activity, arrests telomere loss through addition of the
TTAGGG repeats de novo, to the ends of the chromosome. The telomere/telomerase maintenance
is an inevitable necessity to delay aging and for a healthy lifespan. Here, we report the potential of
full-spectrum, high concentration Ashwagandha (Withania somnifera), an Ayurvedic medicinal
herb, root extract to increase telomerase activity. HeLa cells, when treated with various concentrations
of Ashwagandha root extract, showed an increase in telomerase activity measured with
the established Telomerase Rapid Amplification Protocol (TRAP) assay. Ashwagandha root extract
increased telomerase activity with highest enhancement of ~45% at 10 - 50 μg concentration.
Thus, Ashwagandha root extract has the anti-aging inducing potential.
... First, reserpine's general toxicity was assessed in worms. Reserpines dose-and time-dependently decreased survival in WT N2 and BY200 worms (Fig. 1), corroborating earlier observation (Arya et al. 2009;Saharia et al. 2012;Srivastava et al. 2008;Tauffenberger et al. 2013). Moreover, reserpine delayed the worms' development (Fig. 2), decreased their feeding rate, and altered defecation cycles and egg laying (Table 1). ...
Reserpine is used as an animal model of parkinsonism. We hypothesized that the involuntary movements induced by reserpine in rodents are induced by dopaminergic toxicity caused by extracellular dopamine accumulation. The present study tested the effects of reserpine on the dopaminergic system in Caenorhabditis elegans. Reserpine was toxic to worms (decreased the survival, food intake, development and changed egg laying and defecation cycles). In addition, reserpine increased the worms' locomotor rate on food and decreased dopamine levels. Morphological evaluations of dopaminergic CEP neurons confirmed neurodegeneration characterized by decreased fluorescence intensity and the number of worms with intact CEP neurons, and increased number of shrunken somas per worm. These effects were unrelated to reserpine's effect on decreased expression of the dopamine transporter, dat-1. Interestingly, the locomotor rate on food and the neurodegenerative parameters fully recovered to basal conditions upon reserpine withdrawal. Furthermore, reserpine decreased survival in vesicular monoamine transporter and dat-1 loss-of-function mutant worms. In addition, worms pre-exposed to dopamine followed by exposure to reserpine had decreased survival. Reserpine activated gst-4, which controls a phase II detoxification enzymes downstream of nuclear factor (erythroid-derived-2)-like 2. Our findings establish that the dopamine transporter, dat-1, plays an important role in reserpine toxicity, likely by increasing extracellular dopamine concentrations.
... Arya [3]의 논문에서 C. elegans에 reserpine을 투여한 결과, 비록 Aβ 독성에 의해 유도되는 마비를 완화시키는 특성을 나 타내었으나 Aβ의 축적은 변화 없었고, Aβ oligomer와 mono- mer의 변화도 관찰되지 않았다 [44]. 앞선 결과와는 다르게 본 실험에서는 마우스에 reserpine을 투여한 결과 Aβ-42 peptide 의 농도 및 해마에서의 축적이 감소되었다. ...
Reserpine, an anti-hypertensive drug, is able to positively modulate several phenotypes associated with toxicity in a Caenorhabditis elegans model of Alzheimer's disease (AD). We investigated into the therapeutic effects of reserpine on mammalian neurodegenerative disorders, and found that significant alteration of the key factors influencing AD was detected in Tg2576 mice after reserpine treatment for 30 days. The aggressive behavior of Tg2576 mice was significantly improved upon reserpine treatment, whereas their social contact was consistently maintained. Furthermore, the levels of -42 peptide in the hippocampus of the brain and blood serum were lower in the reserpine-treated group than in the vehicle-treated group. Among g-secretase components, the expression levels of PS-2, Pen-2, and APH-1 were slightly lower in reserpine-treated Tg2576 mice, although a significant change in nicastrin (NCT) expression was not detected. Furthermore, the serum level of nerve growth factor (NGF) increased in reserpine-treated Tg2576 mice compared with vehicle-treated mice. Among down-stream effectors of the NGF receptor TrkA signaling pathway, reserpine treatment induced elevation of TrkA phosphorylation and reduction of ERK phosphorylation. In addition, in the NGF receptor signaling pathway, the expression levels of and Bcl-2 were enhanced in reserpine-treated Tg2576 mice compared with vehicle-treated mice, whereas the expression level of RhoA declined. Overall, these results suggest that reserpine can help relieve AD pathogenesis in Tg2576 mice through downregulation of -42 deposition, alteration of -secretase components, and regulation of NGF metabolism.
... The hlh-17(ns204) animals are not defective in acetylcholine release It is possible that the SWIP response of hlh-17(ns204) animals is insensitive to both reserpine and fluoxetine because HLH-17 influences the activity of C. elegans biogenic amines in a manner that, with the exception of dopamine, does not involve the regulation of genes directly involved in neurotransmitter synthesis, packaging, or transport. A more attractive, alternative possibility is that HLH-17 influences acetylcholine release, as the phenotypic effects of both reserpine (Saharia et al. 2012) and fluoxetine (Bolanos et al. 2002;Chau et al. 2011) are dependent on acetylcholine. In support of this possibility, the inhibitory effect of fluoxetine on acetylcholine release in rats is dependent on activity of the dopaminergic D2 receptors (Bolanos et al. 2002). ...
In Caenorhabditis elegans, the dopamine transporter DAT-1 regulates synaptic dopamine (DA) signaling by controlling extracellular DA levels. In dat-1(ok157) animals, DA is not taken back up presynaptically but instead reaches extrasynpatic sites where it activates the dopamine receptor DOP-3 on choligeneric motor neurons and causes animals to become paralyzed in water. This phenotype is called swimming induced paralysis (SWIP) and is dependent on dat-1 and dop-3. Upstream regulators of dat-1 and dop-3 have yet to be described in C. elegans. In our previous studies, we defined a role for HLH-17 during dopamine response through its regulation of the dopamine receptors. Here we continue our characterization of the effects of HLH-17 on dopamine signaling. Our results suggest that HLH-17 acts downstream of dopamine synthesis to regulate the expression of dop-3 and dat-1. First, we show that hlh-17 animals display a SWIP phenotype that is consistent with its regulation of dop-3 and dat-1. Second, we show that this behavior is enhanced by treatment with the dopamine reuptake inhibitor, bupropion, in both hlh-17 and dat-1 animals, a result suggesting that SWIP behavior is regulated via a mechanism that is both dependent and independent of DAT-1. Third, and finally, we show that although the SWIP phenotype of hlh-17 animals is unresponsive to the dopamine agonist, reserpine, and to the antidepressant, fluoxetine, hlh-17 animals are not defective in acetylcholine signaling. Taken together, our work suggests that HLH-17 is required to maintain normal levels of dopamine in the synaptic cleft through its regulation of dop-3 and dat-1.
... Reserpine blocks vesicular monoamine transporter (VMAT) inhibiting catecholamines and other neurotransmitters from reaching the synapse. In a C. Elgans model, reserpine prolonged lifespan and protected against a-beta neurotoxicity by modulating acetylcholine [226]. Anti-Asthma Drugs (Table 10) Most asthma drugs hold some antiinflammatory activity which has led to studies of their potential benefit in AD. ...
Alzheimer's disease (AD) is the most common cause of dementia and a major cause of morbidity and mortality. The greatest risk factor for AD is age and as many countries are experiencing an aging population, the expected rise in AD threatens to have serious medical and socioeconomic impact in the coming decades. The only approved medications for AD are symptomatic and there are no currently available disease modifying treatments. Hence, a disease modifying treatment is desperately needed for AD not only for proper care and management of affected patients, but also to reduce society socioeconomic burden. Developing novel compounds for any indication is a time, effort, and money consuming endeavor and most treatments never make it to market. Other research and development strategies are needed, especially for the treatment of AD. We provide a review of the current literature in assessing possibilities of repurposing medications currently used for non-AD indications. Many different compounds from many different pharmacological classes have already been studied in an AD context. We provide a pragmatic drug repurposing score for each of these compounds based on type of studies conducted, number of possible mechanisms of action, efficacy in AD and other neurodegenerative disease studies, tolerability profile, and their ability to cross the blood brain barrier. The current data suggest several compounds worthy of further study as treatments for AD. Compounds with the highest scores include lithium, minocycline, exenatide, valproic acid, methylene blue, and nicotine.
... Reserpine, a FDA-approved antihypertensive drug, increases C. elegans lifespan with a high quality of life and ameliorates A toxicity in C. elegans. So far its mode of action and the pathways of activation are not known, but AChE was found to be the crucial player for Reserpine action [51]. ...
Abstract
Alzheimer's disease (AD) is one of the major neurodegenerative diseases affecting almost 28 million people around the globe. It consistently remains one of the major health concerns of present world. Due to the clinical limitations like severe side effects of some synthesized drugs, alternative forms of treatments are gaining global acceptance in the treatment of AD. Neuroprotective compounds of natural origin and their synthetic derivatives exhibit promising results with minimal side effects and some of them are in their different phases of clinical trials. Alkaloids and their synthetic derivatives form one of the groups which have been used in treatment of neurodegenerative diseases like AD. We have further grouped these alkaloids into different sub groups like Indoles, piperdine and isoquinolines. Polyphenols form another important class of natural compounds used in AD management.
... In a subsequent study, this group demonstrated the ability of reserpine to ameliorate the effects of Aβ induced toxicity (Arya et al., 2009) in a C. elegans disease model for Alzheimer's (Link, 1995) and importantly showed that the drug did not merely decrease Aβ levels. In a recent follow-up study, they demonstrated that the modulation of lifespan and Aβ toxicity is related to acetylcholine signaling, as several mutants in acetylcholine signaling either do not respond to reserpine treatment or respond in a negative manner (Saharia et al., 2012). Since acetylcholine signaling is important for feeding behaviors, it seems worthwhile to further evaluate whether resperine is acting via a dietary restriction mechanism to extend lifespan. ...
There is considerable interest in identifying small, drug-like compounds that slow aging in multiple species, particularly in mammals. Such compounds may prove to be useful in treating and retarding age-related disease in humans. Just as invertebrate models have been essential in helping us understand the genetic pathways that control aging, these model organisms are also proving valuable in discovering chemical compounds that influence longevity. The nematode Caenorhabditis elegans has numerous advantages for such studies including its short lifespan and has been exploited by a number of investigators to find compounds that impact aging. Here, we summarize the progress being made in identifying compounds that extend the lifespan of invertebrates, and introduce the challenges we face in translating this research into human therapies.
Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer’s disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.
Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.
Ethnopharmacological relevance:
In traditional Chinese medicine, Shen Nong, BenCao Jing, and Compendium of Materia Medica (Bencao Gangmu), Panax ginseng, and its prescriptions have been used for the treatment of dementia, depression, weight loss, Xiaoke disease (similar to diabetes), and vertigo. All these diseases are associated with the drug-controllable risk factors for Alzheimer's disease (AD), including depression, obesity, diabetes, and hypertension. Ginsenoside Rg1, one of the main active ingredients of P. ginseng and its congener Panax notoginseng, possesses therapeutic potentials against AD and associated diseases. This suggests that ginsenoside Rg1 might have the potential for AD prevention and treatment. Although the anti-AD effects of ginsenoside Rg1 have received more attention, a systematic review of its effects on depression, obesity, diabetes, and hypertension is not available.
Aim of the review:
This systematic literature review comprehensively summarized existing literature on the therapeutic potentials of ginsenoside Rg1 and ginseng in AD prevention and treatment for the propose of providing a foundation of future research aimed at enabling the use of such drugs in clinical practice.
Methods:
Information on ginsenoside Rg1 was collected from relevant published articles identified through a literature search in electronic scientific databases (PubMed, Science Direct, and Google Scholar). The keywords used were "Ginsenoside Rg1," "Panax ginseng," "Source," "Alzheimer's disease," "Brain disorders," "Depression," "Obesity," "Diabetes," and "Hypertension."
Results:
The monomer ginsenoside Rg1 can be relatively easily obtained and has therapeutic potentials against AD. In vitro and in vivo experiments have demonstrated the therapeutic potentials of ginsenoside Rg1 against the drug-controllable risk factors of AD including depression, obesity, diabetes, and hypertension. Thus, ginsenoside Rg1 alleviates diseases resulting from AD risk factors by regulating multiple targets and pathways.
Conclusions:
Ginsenoside Rg1 has the potentials to cure and prevent AD by alleviating depression, obesity, diabetes, and hypertension.
The use of dietary supplements, herbal extracts, and nutraceuticals, of both dietary and nondietary origin, has increased tremendously in the past few decades. As with other chemicals, it is critical to evaluate both their therapeutic potential and safety. Nutraceuticals have been and are continuing to be investigated for their neuroprotective and neurotherapeutic potential, particularly in the context of neurodegenerative diseases. In this chapter, the usefulness of the nematode, Caenorhabditis elegans, as an in vivo, rapid, high-throughput, cost-effective, and efficient model to screen nutraceuticals for their neuroprotective properties is reviewed. The suitability and usefulness of the nematode in molecule-screening studies, with emphasis on the nervous system and transgenic models, is highlighted. Studies utilizing this model, which screened nutraceuticals to evaluate their neuroprotective and neurotherapeutic potential in aging, neurotoxicity, neurodegeneration, and prominent neurodegenerative diseases are reviewed. Strategies to increase the effective use of this model are also discussed.
Glutamate (Glu) and Acetylcholine (ACh), are excitatory neurotransmitters, acting through ionotropic (iR) and metabotropic receptors (mR). Importantly, both neurotransmitters and their signalling are impaired in the prevalent neurodegenerative disease-Alzheimer disease (AD). Glu and its signalling cascade’s influence on ACh-neurotransmission (NT) are sparsely understood. The mGluRs coupled to G-protein signalling acting through PI3K cascade (GrpI) or inhibition of adenylate cyclase–cAMP cascade (GrpII and GrpIII) brings about long-lasting structural/functional changes. These complexities are challenging to decipher. Here, we report that human/mouse mGluRs when compared with their Caenorhabditis elegans homologs, MGL-1-3 showed overall of homology of ∼31-39%. Phylogeneitc analysis revealed homology of MGL-2 to GrpI, MGL-3 with Grp1 &II and GRM6 of GrpIII and MGL-1, a low homology that falls between GrpI & GrpII. Then, alteration of ACh-NT in C. elegans loss-of-function mutants of mgl-1, mgl-2, mgl-3, PI3K (age-1) and iGluR (NMDA)(nmr-1) was estimated by well-established acute aldicarb (Ald), that increases ACh at synapse, and levamisole (Lev) (postsynaptic activation of levamisole sensitive iAChR) induced time-dependent paralysis assays. Surprisingly, all of them were hypersensitive to Ald and Lev compared to wildtype (in percentage), namely, mgl-1 -17, 54; mgl-2 - 7.2, 24; mgl-3 –52, 64; age-1 – 27, 32; nmr-1- 24, 48; respectively. Of the three, mgl-3 contributes to maximal overall acceleration of ACh-NT. Adenylate cyclase, acy-1 gain-of-function mutant showed less hypersensitivity, Ald – 7% and Lev- 25%. Together, Glu receptors and signalling cascades are altering ACh-NT permanently, thus establishing the interplay between them thereby provide potential drug targets to be considered for AD.
Background:
Fructose-common sweetener, consumed in large quantities, is now known to be associated with various metabolic diseases. Recent reports suggest fructose's involvement in neurodegeneration, neurotoxicity, and neuroinflammation. But, its impact at cellular and subcellular level and on energy metabolism, especially, mitochondrial bioenergetics, in neurons is not known.
Objectives:
To study the adverse effects of high fructose in general, and on the mitochondria in a spinal cord motor neuron cell line, NSC-34, in vitro, and Caenorhabditis elegans in vivo.
Methods:
NSC-34 was treated with 0.5%-5% of fructose for different time periods. Fructose's effect on cell viability (MTT assay), metabolic activity (XF24 Seahorse assays) and C. elegans, chronically fed with 5% fructose and alteration in healthspan/mitochondria was monitored.
Results:
In NSC-34: Fructose at 4-5% elicits 60% cell death. Unlike 1%, 5% fructose (F5%) decreased mitochondrial membrane potential by 29%. Shockingly, 6hours F5% treatment almost abolished mitochondrial respiration - basal-respiration (∨123%), maximal-respiration (∨ 95%) and spare-respiratory-capacity (∨ 83%) and ATP production (∨98%) as revealed by XF 24- Seahorse assays. But non - mitochondrial respiration was spared. F5% treatment for 48hrs resulted in the total shutdown of respiratory machinery including glycolysis. Chronic feeding of wildtype C.elegans to F5% throughout, shortened lifespan by ~3 days (∨ 17%), progressively reduced movement (day-2 -∨10.25%, day-5 -∨25% and day-10 -∨56%) and food intake with age (day-5-∨9% and day-10 -∨48%) and instigated mitochondrial swelling and disarray in their arrangement in adult worms body-wall muscle cells.
Conclusion:
Chronic exposure to high fructose negatively impacts cell viability, mitochondrial function, basal glycolysis, and healthspan.
Hedyotis diffusa Willd (Rubiaceae) is a widely used and resourceful traditional Chinese medicine that exerts protection against aging and age-related diseases. However, the underlying mechanisms of the protective effects remain largely unclear. Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which β-amyloid (Aβ)-induced toxicity has been suggested as a main cause. Herein, we use the transgenic Caenorhabditis elegans CL4176, CL2006, and CL2355 strains, which express human Aβ1-42 peptide, to investigate the effects and the possible mechanisms of n-butanol extract of H.diffusa (HDB)-mediated protection against Aβ toxicity in vivo. During the experiments, a method of quality control for HDB was established by HPLC. Additionally, we examined the effects of HBD on gene expression changes with qRT-PCR, aggregation of Aβ plagues with thioflavin-S staining, and protein detection with GFP labeling. HDB improved lifespan, locomotion, and stress resistance. Further study showed that HDB decreased paralysis, the accumulation of ROS, and AChE activity. Moreover, HDB suppressed neuronal Aβ-expression-induced defects in chemotaxis behavior and increased SOD activity. HDB also downregulated the Aβ mRNA level and decreased the number of Aβ deposits. Furthermore, HDB increased the expression levels of sod-3, daf-16, hsf-1, and hsp-16.2 gene and upregulated hsp-16.2::GFP and gst-4::GFP expression. Taken together, these results suggest that HDB may protect against Aβ-induced toxicity in C. elegans via the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway.
Aging is a process characterized by accumulating degenerative damages, resulting in the death of an organism ultimately. The main goal of aging research is to develop therapies that delay age-related diseases in human. Since signaling pathways in aging of Caenorhabditis elegans (C. elegans), fruit flies and mice are evolutionarily conserved, compounds extending lifespan of them by intervening pathways of aging may be useful in treating age-related diseases in human. Natural products have special resource advantage and with few side effect. Recently, many compounds or extracts from natural products slowing aging and extending lifespan have been reported. Here we summarized these compounds or extracts and their mechanisms in increasing longevity of C. elegans or other species, and the prospect in developing anti-aging medicine from natural products.
Neurodegenerative diseases, such as Alzheimer's disease (AD), are multifactorial diseases involving age, genetics, and environment. As such, the model in which one examines disease biology and potential therapeutics should incorporate these three salient features. In both cell culture systems and rodent models, both environment contributions and genetic background can be easily assessed. However, studying the effects of age is either not possible (cell culture) or expensive (rodents), especially for drug discovery. Additionally, the complex mammalian brain is not the easiest to image in vivo for AD pathology hallmarks. For these reasons, simple genetic model organisms can be utilized. Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio have long been used in research; taking advantage of short lifespans, well-characterized genetics, and inexpensive maintenance for environmental and pharmacology studies. In this chapter, the benefits of these organisms for AD research will be discussed, including available transgenics and potential for use in pharmaceutical testing.
Although nutraceutical use goes back millennia, only recently have they been researched systematically. As the number of compounds for study grows, the importance of inexpensive, relevant, and high-throughput methods has increased. Regulatory approval requires using traditional model organisms, but early studies can be conducted in alternative models, such as Caenorhabditis elegans (C. elegans). Claims associated with nutraceuticals often focus on oxidative stress, inflammation protection, and increased longevity. Because many such cellular pathways were originally delineated in C. elegans, this organism could provide researchers with valuable mechanistic data. Two well-described pathways in C. elegans regulating these responses are transforming growth factor-β (TGF-β) and insulin/insulin-like growth factor-1 (IGF-1). Although much simpler in C. elegans than humans, there is homology at each signal transduction step. The goal of this chapter is to emphasize why C. elegans could be used to move mechanistic nutraceutical research forward, particularly in the context of TGF-β and insulin/IGF-1 pathways.
Adverse reports against dichlorvos (DDVP, an organophosphate insecticide) exposure to organisms necessitate studies towards organismal resistance/tolerance by way of pharmacological or genetic means. In the context to genetic modulation, a mutation in methuselah (mth, encodes for a class-II G-protein coupled receptor) is reported to extend (~35%) lifespan of Drosophila melanogaster and enhance their resistance against oxidative stress induced by paraquat exposure (short-term high-level). Lack of studies on organismal tolerance against DDVP by genetic modulation prompted us to examine the protective efficacy of mth mutation in exposed Drosophila. Flies were exposed to 1.5 and 15.0ng/ml of DDVP for 12-48h to examine oxidative stress endpoints and chemical resistance. After prolonged DDVP exposure to flies, anti-oxidant enzyme activities, oxidative stress, glutathione (GSH) content and locomotor performance were assayed at different days (0, 10, 20, 30, 40, 50) of their age. Flies with mth mutation (mth(1)) showed improved chemical resistance and rescued redox-impairments against acute DDVP exposure. Exposed mth(1) flies exhibited improved lifespan along with enhanced anti-oxidant enzyme activities and rescued oxidative perturbations and locomotor insufficiency up to middle age (~20 days) of their life over similarly exposed w(1118) flies. However, at late (>30 days) age, these benefits were undermined. Further, in similarly exposed mth knockdown flies, effects were similar as observed in mth(1) flies. The study provides evidence of tolerance in organism carrying mth mutation against prolonged DDVP exposure and further warrants examination of similar class II GPCR signaling facets towards better organismal health.
Copyright © 2015. Published by Elsevier Inc.
The host-guest interaction between sulfobutylether-β-cyclodextrin (SBE-β-CD) and reserpine (RSP) is described using flow injection-chemiluminescence (FI-CL) and site-directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE-β-CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ∆I = 107.1lgCRES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5-106.1%. Using the proposed CL model, the binding constant (KCD-R ) and the stoichiometric ratio of SBE-β-CD/RSP were calculated to be 7.4 × 10(6) M(-1) and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE-β-CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE-β-CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host-guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.
In this article, we propose very simple procedures to analyze two important indole alkaloids, reserpine and yohimbine, in Rauvolfia yunnanensis. A nonaqueous CE method has been employed using berberine as an internal standard. Optimum separation conditions were obtained when the sample was injected by pressure for 3 s at 50 mbar and then separated with the buffer containing 40 mM ammonium acetate, 2.0% (v/v) acetic acid, and 20% (v/v) acetonitrile in methanol medium at an applied voltage of 25 kV. The analytes were detected at 220 nm. The two alkaloids can be separated within 15 min and quantified with high sensitivity. Good recoveries were obtained with both the procedures, using an internal standard. Finally, these procedures were applied to the analysis of two alkaloids in Rauvolfia yunnanensis.
Recent experimental and industrial advances in the field of nanotechnologies have boosted the development of interdisciplinary research, one of the most constructive and inspiring of human pursuits. Many scientists specialize in manufacturing new forms of nanomaterials that hold promise for various applications such as medical diagnosis and therapy, environmental monitoring, energy production and storage, molecular computing and much more. Graphene, an increasingly important nanosized material reported in 2004, has emerged to become an exciting two-dimensional material with distinct attributes that has attracted great interest in the fields of physics, chemistry, biology and medicine, as well as their related interdisciplinarities. The unique nature of graphene makes it stand out and applicable to various technologies. Its photoelectric properties and inherent Raman spectroscopy make it an ideal candidate for the development of new devices and methods in many branches of life sciences and technology. The electronic properties of graphene make it a highly useful nanomaterial. It carries a high charge mobility which allows it to be extensively applied to field effect transistors. Graphene also has unparalleled transparency and conductivity, making it a viable electrode in solar cells. In addition, the specific area of graphene makes energy storage possible. Although these properties are inherent to the compound, graphene can also be modified into a better nanomaterial. Due to its outstanding performance, graphene development shows great promise in several scientific fields. This paper aims to elaborate on the details of current studies using graphene with regards to the optical and electronic characteristics, fabrication techniques, and various relevant applications.
Alzheimer's disease (AD) and diabetes are 13 among the most common diseases associated with age-14 ing. The pathology of AD is strongly associated with 15 accumulated misfolding proteins that results in neuronal 16 dysfunction within the brain. Diabetes, on the contrary, 17 is characterised by altered insulin signaling that results 18 in reduced glucose uptake, metabolic suppression of 19 energy consuming cells and conversion of glucose to 20 fat in the liver. Despite distinguishing features, these 21 diseases share common elements and may in fact be 22 viewed as fundamentally similar disorders that differ in 23 magnitude of specific traits, primarily affected tissues 24 and time of onset. In this review, we outline the funda-25 mental basis of each of the two diseases and highlight 26 similarities in their pathophysiology. Further ahead we 27 will discuss these features in relation to the develop-28 ment of drugs to treat these two diseases, particularly 29 AD, for which the development of therapeutic chemicals 30 has proven to be particularly difficult. We conclude with 31 comments on efforts to develop a simple organism, 32 Caenorhabditis elegans, as a genetic model to be used 33 to study the systems biology of diabetes and AD.
Alzheimer's disease and inclusion body myositis (IBM) are disorders frequently found in the elderly and characterized by the presence of amyloid-β peptide (Aβ) aggregates. We used Caenorhabditis elegans that express Aβ in muscle cells as a model of IBM, with the aim of analyzing Aβ-induced muscle pathology and evaluating the consequences of modulating Aβ aggregation. First, we tested whether the altered motility we observed in the Aβ transgenic strain could be the result of a compromised neuromuscular synapse. Our pharmacological analyses show that synaptic transmission is defective in our model and suggest a specific defect on nicotine-sensitive acetylcholine receptors (AChRs). Through GFP-coupled protein visualization, we found that synaptic dysfunction correlates with mislocalization of ACR-16, the AChR subunit essential for nicotine-triggered currents. Histological and biochemical analysis allowed us to determine that copper treatment increases the amyloid deposits and decreases Aβ oligomers in this model. Furthermore, copper treatment improves motility, ACR-16 localization, and synaptic function and delays Aβ-induced paralysis. Our results indicate that copper modulates Aβ-induced pathology and suggest that Aβ oligomers are triggering neuromuscular dysfunction. Our findings emphasize the importance of neuromuscular synaptic dysfunction and the relevance of modulating the amyloidogenic component as an alternative therapeutic approach for this debilitating disease.
This paper reviews the pharmacology of Indian medicinal plants, starting with the historical background of European work on the subject beginning as early as the 17th century, and tracing its history through the work of Sen and Bose in the 1930's, and Vakhil's historic 1949 paper on Sarpaghanda. The often crucial role of patient feedback in early discoveries is highlighted, as is the time lag between proof of pharmacological action and identification of the active principle, and subsequent elucidation of mechanism of action. In the case of Indian plants in the 20th century this process sometimes took almost 50 years. Reserpine and its mechanisms are given in detail, and its current relevance to public health discussed. The foundation of present day methods of pharmacology is briefly presented so the complexity of methods used to identify properties of Ayurveda derived drugs like forskolin and baicalein, and their bioavailability, may be better appreciated. Ayurveda derived anti-oxidants and their levels of action, immuno-modulators, particularly with respect to the NF-kB pathway and its implications for cancer control, are all considered. The example of curcumin derived from turmeric is explained in more detail, because of its role in cancer prevention. Finally, the paper emphasizes the importance of Ayurveda's concepts of rasayana as a form of dietary chemo-prevention; the significance of ahar, diet, in Ayurveda's aspiration to prevent disease and restore health thus becomes clear. Understood in this light, Ayurveda may transcend pharmacology as a treatment paradigm.
Our previous work indicated that novel analogs of choline have cytoprotective effects in vitro that might be useful in neurodegenerative conditions such as Alzheimer's disease (AD). Furthermore, two lead compounds (JWB1-84-1 and JAY2-22-33) from a library of more than 50 improved cognitive performances in a transgenic mouse model of AD. The purpose of these experiments was to more specifically investigate the neuroprotective capabilities of these lead compounds both in vitro and in vivo.
We used N2a cells which express a Swedish mutation in the amyloid precursor protein and presenilin 1 genes to investigate the effect of JWB1-84-1 and JAY2-22-33 on β-amyloid (Aβ) levels and found that both compounds significantly reduced Aβ levels. JWB1-84-1 and JAY2-22-33 also protected rat primary cortical neurons from Aβ toxicity. Subsequently, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism to identify potential molecular targets of these compounds. In the C. elegans model of Aβ toxicity, human Aβ is expressed intracellularly in the body wall muscle. The expression and subsequent aggregation of Aβ in the muscle leads to progressive paralysis.
We found that JAY2-22-33 (but not JWB1-84-1) significantly reduced Aβ toxicity by delaying paralysis and this protective effect required both the insulin signaling pathway and nicotinic acetylcholine receptors (nAChRs).
Defining a behavior that requires the function of specific neurons in the free-living nematode Caenorhabditis elegans can allow one to screen for mutations that disrupt the specification or function of those neurons. We identified serotonin-immunoreactive neurons required for tail curling or "turning" behavior exhibited by C. elegans males during mating. Males mutant in three different genes that reduce serotonin expression, cat-1, cat-4, and bas-1, exhibited defects in turning behavior similar to those of wild-type males in which these neurons were ablated. The turning defect of cat-4 males was rescued by exogenous serotonin, consistent with the idea that their behavioral defect is caused by a lack of serotonin. While the serotonin-deficient mutants we analyzed shared certain behavioral traits, they were blocked for serotonin synthesis at different steps. Analysis of these and additional serotonin-deficient mutants may help us understand how a neuron controls the expression of a serotonergic phenotype.
age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20 degrees; at 25 degrees age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites). Mutant males also show extended life spans. age-1(hx546) is associated with a 75% decrease in hermaphrodite self-fertility as compared to the age-1+ allele at 20 degrees. Using two novel strategies for following the segregation of age-1, we present evidence that longer life results from a mutation in a single gene that increases the probability of survival at all chronological ages. The long-life and reduced-fertility phenotypes cosegregate and are tightly linked to fer-15, a locus on linkage group II. age-1(hx546) does not affect the timing of larval molts, the length of embryogenesis, food uptake, movement, or behavior in any way tested. Although age-1(hx546) lowers hermaphrodite self-fertility, it does not markedly affect the length of the reproductive period with all the increase in life expectancy due to an increase in the length of postreproductive life. In so far as we are aware, this mutant in age-1 is the only instance of a well-characterized genetic locus in which the mutant form results in lengthened fife. It is likely that the action of age-1 in lengthening life results not from eliminating a programmed aging function but rather from reduced hermaphrodite self-fertility or from some other unknown metabolic or physiologic alteration.
A C. elegans neurosecretory signaling system regulates whether animals enter the reproductive life cycle or arrest development at the
long-lived dauer diapause stage. daf-2, a key gene in the genetic pathway that mediates this endocrine signaling, encodes an insulin receptor family member. Decreases
in DAF-2 signaling induce metabolic and developmental changes, as in mammalian metabolic control by the insulin receptor.
Decreased DAF-2 signaling also causes an increase in life-span. Life-span regulation by insulin-like metabolic control is
analogous to mammalian longevity enhancement induced by caloric restriction, suggesting a general link between metabolism,
diapause, and longevity.
The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced
activity of the genedaf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as
long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2mutations requires the activity of the gene daf-16. daf-16appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead
family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the
activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved.
We have identified the Caenorhabditis elegans homolog of the mammalian vesicular monoamine transporters (VMATs); it is 47% identical to human VMAT1 and 49% identical to human VMAT2. C. elegans VMAT is associated with synaptic vesicles in approximately 25 neurons, including all of the cells reported to contain dopamine and serotonin, plus a few others. When C. elegans VMAT is expressed in mammalian cells, it has serotonin and dopamine transport activity; norepinephrine, tyramine, octopamine, and histamine also have high affinity for the transporter. The pharmacological profile of C. elegans VMAT is closer to mammalian VMAT2 than VMAT1. The C. elegans VMAT gene is cat-1; cat-1 knock-outs are totally deficient for VMAT immunostaining and for dopamine-mediated sensory behaviors, yet they are viable and grow relatively well. The cat-1 mutant phenotypes can be rescued by C. elegans VMAT constructs and also (at least partially) by human VMAT1 or VMAT2 transgenes. It therefore appears that the function of amine neurotransmitters can be completely dependent on their loading into synaptic vesicles.
In the nematode Caenorhabditis elegans, an insulin receptor signaling pathway regulates adult life span and developmental arrest at the dauer larval stage. Here we show that the unc-64 and unc-31 genes also function in this pathway. These two genes are involved in mediating Ca2+-regulated secretion. Mutations in unc-64 and unc-31 increase adult life span and cause constitutive dauer formation. Both phenotypes are suppressed by mutations in daf-16, which also suppresses other mutations in this pathway. We present evidence that the site of action of unc-64 is neuronal, suggesting that a neurosecretory signal regulates life span and dauer formation.
Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. We measured evoked postsynaptic currents at the neuromuscular junction in both wild-type and mutants and observed that the removal of SLO-1 greatly increased quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release.
We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.
Aging is characterized by progressive, degenerative changes in many tissues. To elucidate the relationships among degenerative changes in Caenorhabditis elegans, we developed methods to measure age-related changes quantitatively and analyzed correlations among these changes by using a longitudinal study. The age-related declines of pharyngeal pumping and body movement were positively correlated with each other and lifespan. These findings suggest that the declines of pharyngeal pumping and body movement cause a decline in survival probability or that a shared regulatory system mediates the declines in pharyngeal pumping, body movement, and survival probability. Furthermore, measurements of these processes can be used to predict lifespan and detect premature aging. The declines of physiological processes were measured in daf-2, age-1, daf-16, eat-2, and clk-1 mutants that have altered lifespans. Each mutant strain displayed changes in one or more age-related declines, but the correlations among age-related changes were similar to WT. These measurements were used to generate a system of four stages that describes the aging process and is useful for the analysis of genetic and environmental effects on aging.
Intracellular amyloidbeta peptide (iAbeta1-42) accumulates in the Alzheimer's disease brain before plaque and tangle formation (Gouras et al., 2000) and is extremely toxic to human neurons (Zhang et al., 2002). Here, we investigated whether androgen and estrogen could prevent iAbeta1-4) toxicity, because both these hormones have a wide range of neuroprotective actions. At physiological concentrations, 17-beta-estradiol, testosterone, and methyl testosterone reduce iAbeta1-42-induced cell death by 50% in neurons treated after the injection and by 80-90% in neurons treated 1 hr before the injection. The neuroprotective action of the hormones is mediated by receptors, because the estrogen receptor (ER) antagonist tamoxifen and the androgen receptor (AR) antagonist flutamide completely block the estrogen- and androgen-mediated neuroprotection, respectively. Transcriptional activity is required for the neuroprotective action, because dominant negative forms of the receptors that block the transcriptional activity of the ER and AR prevent estrogen- and androgen-mediated neuroprotection. Proteomics followed by Western blot analyses identified increased levels of heat shock protein 70 (Hsp70) in testosterone- and estrogen-treated human neurons. Comicroinjection of Hsp70 with the iAbeta1-42 blocks the toxicity of iAbeta1-42. We conclude that estrogen and androgens protect human neurons against iAbeta1-42 toxicity by increasing the levels of Hsp70 in the neurons.
Caloric restriction extends lifespan in numerous species. In the budding yeast Saccharomyces cerevisiae this effect requires Sir2 (ref. 1), a member of the sirtuin family of NAD+-dependent deacetylases. Sirtuin activating compounds (STACs) can promote the survival of human cells and extend the replicative lifespan of yeast. Here we show that resveratrol and other STACs activate sirtuins from Caenorhabditis elegans and Drosophila melanogaster, and extend the lifespan of these animals without reducing fecundity. Lifespan extension is dependent on functional Sir2, and is not observed when nutrients are restricted. Together these data indicate that STACs slow metazoan ageing by mechanisms that may be related to caloric restriction.
The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR alpha subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two alpha types (UNC-38 and UNC-63) and two non-alpha types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.
The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: gamma-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the gamma-aminobutyric acid- and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the approximately 40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor alpha7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisole-sensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.
The Caenorhabditis elegans transcription factor HSF-1, which regulates the heat-shock response, also influences aging. Reducing hsf-1 activity accelerates tissue aging and shortens life-span, and we show that hsf-1 overexpression extends lifespan. We find that HSF-1, like the transcription factor DAF-16, is required for daf-2–insulin/IGF-1 receptor mutations to extend life-span. Our findings suggest this is because HSF-1 and DAF-16 together activate
expression of specific genes, including genes encoding small heat-shock proteins, which in turn promote longevity. The small
heat-shock proteins also delay the onset of polyglutamine-expansion protein aggregation, suggesting that these proteins couple
the normal aging process to this type of age-related disease.
The pathogenesis of Alzheimer's disease (AD) has been linked to a deficiency in the brain neurotransmitter acetylcholine. Subsequently, acetylcholinesterase inhibitors (AChEIs) were introduced for the symptomatic treatment of AD. The prevailing view has been that the efficacy of AChEIs is attained through their augmentation of acetylcholine-medicated neuron to neuron transmission. However, AChEIs also protect cells from free radical toxicity and beta-amyloid-induced injury, and increased production of antioxidants. In addition, it has been reported that AChEIs directly inhibit the release of cytokines from microglia and monocytes. These observations are supported by evidence showing a role for acetylcholine in suppression of cytokine release through a 'cholinergic anti-inflammatory pathway'. Based on the accumulating research data so far, it is no longer appropriate to consider that the sole action of AChEIs in AD is through direct acetylcholine-medicated enhancement of neuronal transmission. Evidence points to a possible anti-inflammatory role for these agents as well.
Aberrant protein aggregation is a common feature of late-onset neurodegenerative diseases, including Alzheimer's disease,
which is associated with the misassembly of the Aβ1-42 peptide. Aggregation-mediated Aβ1-42 toxicity was reduced in Caenorhabiditis elegans when aging was slowed by decreased insulin/insulin growth factor–1–like signaling (IIS). The downstream transcription factors,
heat shock factor 1, and DAF-16 regulate opposing disaggregation and aggregation activities to promote cellular survival in
response to constitutive toxic protein aggregation. Because the IIS pathway is central to the regulation of longevity and
youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated
proteotoxicity.
Nicotine has a therapeutic benefit in treating Alzheimer's disease (AD). In the present study we show that nicotine decreases accumulation of beta-amyloid (Abeta) in the cortex and hippocampus of APP (V717I) transgenic mice. Nicotine prevents activation of NF-kappaB and c-Myc by inhibiting the activation of MAP kinases (MAPKs). As a result, the activity of inducible NOS and the production of NO are down-regulated. RNA interference experiments show that the above nicotine-mediated process requires alpha7 nAChR. Nicotine decreases Abeta via the activation of alpha7nAChRs through MAPK, NF-kappaB, and c-myc pathways. Nicotine also inhibits apoptosis and cell cycle progression in this mouse line. The dissected signaling pathway of nicotine-mediated neuroprotection in the present study provides a mechanistic basis for the potential development of drug targets for treating AD.
In recent years, it has become increasingly clear that mitochondrial dysfunction and oxidative damage are major contributors to neuronal loss. Free radicals, typically generated from mitochondrial respiration, cause oxidative damage of nucleic acids, lipids, carbohydrates and proteins. Despite enormous amount of effort, however, the mechanism by which oxidative damage causes neuronal death is not well understood. Emerging data from a number of neurodegenerative diseases suggest that there may be common features of toxicity that are related to oxidative damage. In this review, while focusing on Huntington's disease (HD), we discuss similarities among HD, Friedreich ataxia and xeroderma pigmentosum, which provide insight into shared mechanisms of neuronal death.
Caenorhabditis elegans has emerged as a powerful model system for studying the biology of the synapse. Here we describe a widely used assay for synaptic transmission at the C. elegans neuromuscular junction. This protocol monitors the sensitivity of C. elegans to the paralyzing affects of an acetylcholinesterase inhibitor, aldicarb. Briefly, adult worms are incubated in the presence of aldicarb and scored for the time-course of aldicarb-induced paralysis. Animals harboring mutations in genes that affect synaptic transmission generally exhibit a change in their sensitivity to aldicarb (either increased sensitivity for enhancements in synaptic transmission or decreased sensitivity for blockage in synaptic transmission). This technique provides a simple assay for the accurate comparative analysis of synaptic transmission in multiple C. elegans strains. The protocol described can be performed relatively quickly and is a practical alternative to other techniques used to study synaptic transmission. This protocol can also be modified to follow the paralytic effects with other pharmacological reagents. The assay can be performed in about 3-6 hours depending on the severity of synaptic transmission defects.
The neurotransmitter serotonin has been implicated in affecting the variation of longevity in natural Drosophila populations and age-related diseases in mammals. Based on these observations, it has been predicted that serotonin signal, perhaps at levels of serotonin biosynthesis, may control lifespan. Here, we investigated a variety of mutations in serotonin-signal genes, including serotonin biosynthesis genes, a serotonin transporter gene, and serotonin receptor genes. Despite this prediction, mutations in the serotonin biosynthesis genes had little or modest effects on lifespan, while the mod-5 mutation with increased availability of serotonin caused a modest life-shortening effect. In contrast, a deletion mutation of the ser-1 serotonin receptor gene increased longevity by up to 46%, likely through the insulin/insulin-like growth factor 1 pathway. This result suggests an interaction between the serotonin pathway and the insulin/insulin-like growth factor 1 pathway. A deletion mutation of another serotonin receptor gene, ser-4, shortened early to mid lifespan. The results suggest that serotonin signal antagonistically modulates longevity through different serotonin receptors. This study may indicate serotonin receptors as a potential target for antigeric interventions.
The nematode Caenorhabditis elegans ages and dies in a few weeks, but humans can live for 100 years or more. Assuming that the ancestor we share with nematodes aged rapidly, this means that over evolutionary time mutations have increased lifespan more than 2,000-fold. Which genes can extend lifespan? Can we augment their activities and live even longer? After centuries of wistful poetry and wild imagination, we are now getting answers, often unexpected ones, to these fundamental questions.
The ability of Caenorhabditis elegans to recover from exposure to high doses of aldicarb and fenamiphos was examined at the organismal and biochemical levels by determination of movement and acetylcholinesterase activity. Nematodes recovered rapidly from a 24-hour exposure to both compounds at concentrations that caused complete paralysis. Acetylcholinesterase regained nearly full activity after a 24-hour exposure to aldicarb but only 10% activity after exposure to fenamiphos. The nematodes were able to move normally, however, on the limited activity that was regained after fenamiphos treatment. Mutant C. elegans strains deficient in various molecular forms of acetylcholinesterase were utilized to demonstrate that the mechanism of recovery did not involve new synthesis of enzyme. This result was confirmed by experiments on acetylcholinesterase reactivation from live versus dead nematodes.
Earlier we have reported that reserpine, an antihypertensive drug, known to downregulate biogenic amines through inhibition of the vesicular monoamine transporter (VMAT), increases longevity of Caenorhabditis elegans with a high quality of life, namely, enhanced and prolonged mobility (Srivastava et al., 2008). As neurodegenerative diseases are of adult onset, we addressed the protective ability of reserpine against neurodegenerative diseases, especially Alzheimer's disease (AD). In the well established AD model in C. elegans, Amyloid beta (Abeta) is expressed in the muscles and Abeta toxicity is manifested as paralysis (Link, 1995). In this model, reserpine significantly delayed paralysis and increased the longevity. In addition, reserpine provided thermotolerance, but interestingly the Abeta transcript and expression levels remains grossly unchanged.
Dopamine is the putative transmitter of eight neurons in the hermaphrodite form of the nematode Caenorhabditis elegans. These include the cephalic and deirid neurons, which are believed to be mechanosensory. The male has an additional six dopaminergic neurons in the tail. Mutants have been selected which have defects in the formaldehyde induced fluorescence and lack dopamine to varying degrees, but they are not insensitive to touch. The dopaminergic neurons of C. elegans are compared with the homologous neurons in Ascaris lumbricoides.
unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.
Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of greater than 98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered Km values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene.
We have characterized a small group of genes (13 loci) in the nematode Caenorhabditis elegans that, when mutated, confer resistance to the potent anthelmintic levamisole. Mutants at the 7 loci conferring the most extreme resistance generally possess almost identical visible and pharmacological phenotypes: uncoordinated motor behavior, most severe in early larval life, extreme resistance to cholinergic agonists and sensitivity to hypo-osmotic shock. Mutants with exceptional phenotypes suggest possible functions for several of the resistance loci. The most extreme mutants can readily be selected by their drug resistance (211 mutants, as many as 74 alleles of one gene). The more common resistance loci are likely to be unessential genes, while loci identified by only a few alleles may be essential genes or genes conferring resistance only when mutated in a special way. We propose that these mutants represent a favorable system for understanding how a small group of related genes function in a simple animal. The extreme drug resistance of these mutants makes them useful tools for the genetic manipulation of C. elegans. And, as the most resistant class of mutants might lack pharmacologically functional acetyl-choline receptors (LEWIS et al. 1980), these mutants may also be of some neurobiological significance.
We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.
Transgenic Caenorhabditis elegans nematodes have been engineered to express potentially amyloidic human proteins. These animals contain constructs in which the muscle-specific unc-54 promoter/enhancer of C. elegans drives the expression of the appropriate coding regions derived from human cDNA clones. Animals containing constructs expressing the 42-amino acid beta-amyloid peptide (derived from human amyloid precursor protein cDNA) produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies. A subset of these deposits also bind the amyloid-specific dye thioflavin S, indicating that these deposits have the tinctural characteristics of classic amyloid. Co-expression of beta-peptide and transthyretin, a protein implicated in preventing the formation of insoluble beta-amyloid, leads to a dramatic reduction in the number of dye-reactive deposits. These results suggest that this invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.
We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. Both genes also regulate formation of the dauer larva, a developmentally arrested larval form that is induced by crowding and starvation and is very long-lived. Our findings raise the possibility that the longevity of the dauer is not simply a consequence of its arrested growth, but instead results from a regulated lifespan extension mechanism that can be uncoupled from other aspects of dauer formation. daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.
Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.
We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63-64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.
Low caloric intake (caloric restriction) can lengthen the life span of a wide range of animals and possibly even of humans. To understand better how caloric restriction lengthens life span, we used genetic methods and criteria to investigate its mechanism of action in the nematode Caenorhabditis elegans. Mutations in many genes (eat genes) result in partial starvation of the worm by disrupting the function of the pharynx, the feeding organ. We found that most eat mutations significantly lengthen life span (by up to 50%). In C. elegans, mutations in a number of other genes that can extend life span have been found. Two genetically distinct mechanisms of life span extension are known: a mechanism involving genes that regulate dauer formation (age-1, daf-2, daf-16, and daf-28) and a mechanism involving genes that affect the rate of development and behavior (clk-1, clk-2, clk-3, and gro-1). We find that the long life of eat-2 mutants does not require the activity of DAF-16 and that eat-2; daf-2 double mutants live even longer than extremely long-lived daf-2 mutants. These findings demonstrate that food restriction lengthens life span by a mechanism distinct from that of dauer-formation mutants. In contrast, we find that food restriction does not further increase the life span of long-lived clk-1 mutants, suggesting that clk-1 and caloric restriction affect similar processes.
A large and diverse number of diseases are now recognized as `conformational
diseases', caused by adoption of non-native protein conformations that lead
to aggregation. The recent conference, `Alpha1-antitrypsin deficiency
and other conformational diseases', held in Airlie, Virginia, USA (27–30
June, 2000) focused on some of the common pathways by which cells protect
themselves from toxicity associated with protein misfolding and aggregation.
Evolutionary models of aging propose that a trade-off exists between the resources an organism devotes to reproduction and growth and those devoted to cellular maintenance and repair, such that an optimal life history always entails an imperfect ability to resist stress. Yet, since environmental stressors, such as caloric restriction or exposure to mild stress, can increase stress resistance and life span, it is possible that a common genetic mechanism could regulate the allocation of resources in response to a changing environment (for overview, see ). Consistent with predictions of evolutionary trade-off models, we show that nematodes carrying an integrated DAF-16::GFP transgene grow and reproduce more slowly yet are more stress resistant and longer lived than controls carrying the integration marker alone. We also show that the nuclear localization of the DAF-16::GFP fusion protein responds to environmental inputs as well as genetic. Environmental stresses, such as starvation, heat, and oxidative stress, cause rapid nuclear localization of DAF-16. In conditions rich in food, we find that DAF-16::GFP is inhibited from entry into the nucleus by daf-2 and akt-1/akt-2, both components of insulin-like signaling in nematodes. We suggest that changes in the subcellular localization of DAF-16 by environmental cues allows for rapid reallocation of resources in response to a changing environment at all stages of life.
In C. elegans, the transcription factor DAF-16 promotes longevity in response to reduced insulin/IGF-1 signaling or germline ablation. In this study, we have asked how different tissues interact to specify the lifespan of the animal. We find that several tissues act as signaling centers. In particular, DAF-16 activity in the intestine, which is also the animal's adipose tissue, completely restores the longevity of daf-16(-) germline-deficient animals, and increases the lifespans of daf-16(-) insulin/IGF-1-pathway mutants substantially. Our findings indicate that DAF-16 may control two types of downstream signals: DAF-16 activity in signaling cells upregulates DAF-16 in specific responding tissues, possibly via regulation of insulin-like peptides, and also evokes DAF-16-independent responses. We suggest that this network of tissue interactions and feedback regulation allows the tissues to equilibrate and fine-tune their expression of downstream genes, which, in turn, coordinates their rates of aging within the animal.
Human diseases characterized by insoluble extracellular deposits of proteins have been recognized for almost two centuries. Such amyloidoses were once thought to represent arcane secondary phenomena of questionable pathogenic significance. But it is has now become clear that many different proteins can misfold and form extracellular or intracellular aggregates that initiate profound cellular dysfunction. Particularly challenging examples of such disorders occur in the post-mitotic environment of the neuron and include Alzheimer's and Parkinson's diseases. Understanding some of the principles of protein folding has helped to explain how such diseases arise, with attendant therapeutic insights.
Although Charcot described amyotrophic lateral sclerosis (ALS) more than 130 years ago, the mechanism underlying the characteristic selective degeneration and death of motor neurons in this common adult motor neuron disease has remained a mystery. There is no effective remedy for this progressive, fatal disorder. Modern genetics has now identified mutations in one gene [Cu/Zn superoxide dismutase (SOD1)] as a primary cause and implicated others [encoding neurofilaments, cytoplasmic dynein and its processivity factor dynactin, and vascular endothelial growth factor (VEGF)] as contributors to, or causes of, motor neuron diseases. These insights have enabled development of model systems to test hypotheses of disease mechanism and potential therapies. Along with errors in the handling of synaptic glutamate and the potential excitotoxic response this provokes, these model systems highlight the involvement of nonneuronal cells in disease progression and provide new therapeutic strategies.
Drugs that target the serotonergic system are the most commonly prescribed therapeutic agents and are used for treatment of a wide range of behavioral and neurological disorders. However, the mechanism of the drug action remain a conjecture. Here, we dissect the genetic targets of serotonin (5HT), the selective 5HT reuptake inhibitor (SSRI) fluoxetine (Prozac), the tricyclic antidepressant imipramine, and dopamine. Using the well-established serotonergic response in C. elegans egg-laying behavior as a paradigm, we show that action of fluoxetine and imipramine at the 5HT reuptake transporter (SERT) and at 5HT receptors are separable mechanisms. Even mutants completely lacking 5HT or SERT can partially respond to fluoxetine and imipramine. Furthermore, distinct mechanisms for each drug can be recognized to mediate these responses. Deletion of SER-1, a 5HT1 receptor, abolishes the response to 5HT but has only a minor effect on the response to imipramine and no effect on the response to fluoxetine. In contrast, deletion of SER-4, a 5HT2 receptor, confers significant resistance to imipramine while leaving the responses to 5HT or fluoxetine intact. Further, fluoxetine can stimulate egg laying via the Gq protein EGL-30, independent of SER-1, SER-4, or 5HT. We also show that dopamine antagonizes the 5HT action via the 5HT-gated ion channel MOD-1 signaling, suggesting that this channel activity couples 5HT and dopamine signaling. These results suggest that the actions of these drugs at specific receptor subtypes could determine their therapeutic efficacy. SSRIs and tricyclic antidepressants may regulate 5HT outputs independently of synaptic levels of 5HT.
In chronic neurodegenerative diseases, microglial activation is an early sign that often precedes neuronal death. Increasing evidence indicates that in these chronic pathologies activated microglia sustain a local inflammatory response. Nonetheless, the potential detrimental or protective roles of such reaction remain to date not fully understood, mainly because of the lack of direct evidence of the functional properties acquired by microglia in the course of chronic diseases. Purified microglial cultures have been extensively used to investigate microglial functions associated with activation, but they are often criticized for some experimental constrains, including the abrupt addition of activators, the limited time of stimulation, and the absence of interactions with neurons or other elements of brain parenchyma. To limit these confounding factors, we developed in vitro models in which microglial cells were repeatedly challenged with lipopolysaccharide or co-cultured with healthy, apoptotic, or necrotic neuronal cells. We found that chronic stimulation and interaction with phosphatidylserine-expressing apoptotic cells induced microglial cells to release immunoregulatory and neuroprotective agents (prostaglandin E(2), transforming growth factor-beta, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-alpha and nitric oxide) was inhibited. These findings suggest that signals that are relevant to chronic diseases lead to a progressive down-regulation of pro-inflammatory microglial functions and may help in understanding the atypical microglial activation that begins to be recognized in some chronic neuropathologies.
Alzheimer's disease (AD) has been associated with aggregation of beta-amyloid peptide (Abeta) and cell death in the brain. Using various models, such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the mouse Mus musculus, investigators have attempted to imitate the pathology process of AD for better understanding of the cellular mechanisms and for possible therapeutic intervention. Among many in vitro and in vivo models of AD, transgenic C. elegans expressing human Abeta has shown its own advantages. The transgenic C. elegans model have been used in studying AD due to its short life span, facility to maintain, ability to develop muscle-associated deposits reactive to amyloid-specific dyes and the concomitant progressive paralysis phenotype. Moreover, the transgenic C. elegans exhibits increased levels of reactive oxygen species (ROS) and protein carbonyls, similar to those observed in AD patients, supporting the current theory on Abeta-induced oxidative stress and subsequent neurodegeneration in AD. DNA microarray assays of the worm demonstrated several stress-related genes being upregulated, particularly two genes homologous to human alphaB-crystallin and tumor necrosis factor-related protein, which were also upregulated in postmortem AD brain. Studies in our laboratory along with others suggest that the transgenic C. elegans model is a suitable in vivo model to relate Abeta-expression with its toxicity, which may underlie AD pathology. It may also be used as a tool for pharmacological evaluation of novel therapeutic agents.