Frontiers in Molecular Neuroscience

Published by Frontiers
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Schematic representation of different direct molecular targets of lithium. These include inositol monophosphatases (IMPAs), Bisphosphate 3′-nucleotidase (BPNT), cyclooxygenase (COX), beta-arrestin 2 (βArr2), and members of the glycogen synthase kinase 3 alpha and beta (GSK3α and β). Since the mechanism by which lithium would inhibit several of these molecules is by competing with magnesium ions (Mg2+) acting as a co-factor we also included the possibility that other molecular targets may be affected through such competition.
Schematic representation of signaling pathways regulating the activity of brain GSK3 and its regulation by lithium. Activation of different cell surface receptors activates Phosphatidylinositol 3-kinases (PI3K) that in turn phosphorylates Phosphatidylinositol 4,5-bisphosphate (PIP2) into Phosphatidylinositol (3,4,5)-triphosphate (PIP3). Availability of PIP3 causes the co-recruitment of the 3-phosphoinositide dependent protein kinase-1 (PDK1) and Akt to the cell membrane and the activation of Akt by PDK1. Phosphorylation of N-terminal serine residues of glycogen synthase kinase 3 (GSK3) isoforms by activated Akt results in GSK3 inactivation. Conversely, activation of the D2 dopamine receptor (D2R) and potentially of other G protein coupled receptors (GPCR) triggers the formation of a signaling complex composed of Akt, beta-arrestin 2 (βArr2), and protein phosphatase 2A (PP2A) that results in an inactivation of Akt and concomitant relived GSK3 inhibition. Lithium can affect the equilibrium of this signaling network by inhibiting GSK3 directly and by disrupting the assembly of the Akt;βArr2;PP2A signaling complex. In addition, activated GSK3 would contribute to its own regulation by Akt by stabilizing the formation of this same protein complex.
For more than 60 years, the mood stabilizer lithium has been used alone or in combination for the treatment of bipolar disorder, schizophrenia, depression, and other mental illnesses. Despite this long history, the molecular mechanisms trough which lithium regulates behavior are still poorly understood. Among several targets, lithium has been shown to directly inhibit glycogen synthase kinase 3 alpha and beta (GSK3α and GSK3β). However in vivo, lithium also inhibits GSK3 by regulating other mechanisms like the formation of a signaling complex comprised of beta-arrestin 2 (βArr2) and Akt. Here, we provide an overview of in vivo evidence supporting a role for inhibition of GSK3 in some behavioral effects of lithium. We also explore how regulation of GSK3 by lithium within a signaling network involving several molecular targets and cell surface receptors [e.g., G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs)] may provide cues to its relative pharmacological selectivity and its effects on disease mechanisms. A better understanding of these intricate actions of lithium at a systems level may allow the rational development of better mood stabilizer drugs with enhanced selectivity, efficacy, and lesser side effects.
 
At least three forms of signaling between pre- and postsynaptic partners are necessary during synapse formation. First, "targeting" signals instruct presynaptic axons to recognize and adhere to the correct portion of a postsynaptic target cell. Second, trans-synaptic "organizing" signals induce differentiation in their synaptic partner so that each side of the synapse is specialized for synaptic transmission. Finally, in many regions of the nervous system an excess of synapses are initially formed, therefore "refinement" signals must either stabilize or destabilize the synapse to reinforce or eliminate connections, respectively. Because of both their importance in processing visual information and their accessibility, retinogeniculate synapses have served as a model for studying synaptic development. Molecular signals that drive retinogeniculate "targeting" and "refinement" have been identified, however, little is known about what "organizing" cues are necessary for the differentiation of retinal axons into presynaptic terminals. To identify such "organizing" cues, we used microarray analysis to assess whether any target-derived "synaptic organizers" were enriched in the mouse dorsal lateral geniculate nucleus (dLGN) during retinogeniculate synapse formation. One candidate "organizing" molecule enriched in perinatal dLGN was FGF22, a secreted cue that induces the formation of excitatory nerve terminals in muscle, hippocampus, and cerebellum. In FGF22 knockout mice, the development of retinal terminals in dLGN was impaired. Thus, FGF22 is an important "organizing" cue for the timely development of retinogeniculate synapses.
 
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Dysregulation of microRNA biogenesis in 22q11.2DS animal models. The MIR185 and the DGCR8 genes are located within the minimal 1.5 Mb microdeletion region on chromosome 22q11.2 and the equivalent region of mouse chromosome 16. The microdeletion leads to a hemizygosity of MIR185 and DGCR8. The heterozygous Dgcr8 deficiency is responsible for the reduced biogenesis of a specific subset of microRNAs (red) observed in Df(16)A+/- mice (Stark et al., 2008). These down-regulated microRNAs include miR-185 (black). The pronounced reduction of miR-185 expression (indicated by a broader arrow) may be due to a combined effect of the hemizygosity of the MIR185 gene and the impaired maturation of the pri-miR-185 transcript secondary to reduced Dgcr8 levels (Xu et al., 2013). The resulting alterations in mature microRNA expression levels may lead to altered gene expression of target genes, which might produce some of the neural, cognitive, and behavioral deficits observed in 22q11.2DS. miRISC, microRNA-induced silencing complex.
The 22q11.2 deletion is the strongest known genetic risk factor for schizophrenia. Research has implicated microRNA-mediated dysregulation in 22q11.2 deletion syndrome (22q11.2DS) schizophrenia-risk. Primary candidate genes are DGCR8 (DiGeorge syndrome critical region gene 8), which encodes a component of the microprocessor complex essential for microRNA biogenesis, and MIR185, which encodes microRNA 185. Mouse models of 22q11.2DS have demonstrated alterations in brain microRNA biogenesis, and that DGCR8 haploinsufficiency may contribute to these alterations, e.g., via down-regulation of a specific microRNA subset. miR-185 was the top-scoring down-regulated microRNA in both the prefrontal cortex and the hippocampus, brain areas which are the key foci of schizophrenia research. This reduction in miR-185 expression contributed to dendritic and spine development deficits in hippocampal neurons. In addition, miR-185 has two validated targets (RhoA, Cdc42), both of which have been associated with altered expression levels in schizophrenia. These combined data support the involvement of miR-185 and its down-stream pathways in schizophrenia. This review summarizes evidence implicating microRNA-mediated dysregulation in schizophrenia in both 22q11.2DS-related and idiopathic cases.
 
Proteasome-mediated proteolysis is important for synaptic plasticity, neuronal development, protein quality control, and many other processes in neurons. To define proteasome composition in brain, we affinity purified 26S proteasomes from cytosolic and synaptic compartments of the rat cortex. Using tandem mass spectrometry, we identified the standard 26S subunits and a set of 28 proteasome-interacting proteins that associated substoichiometrically and may serve as regulators or cofactors. This set differed from those in other tissues and we also found several proteins that associated only with either the cytosolic or the synaptic proteasome. The latter included the ubiquitin-binding factor TAX1BP1 and synaptic vesicle protein SNAP-25. Native gel electrophoresis revealed a higher proportion of doubly-capped 26S proteasome (19S-20S-19S) in the cortex than in the liver or kidney. To investigate the interplay between proteasome regulation and synaptic plasticity, we exposed cultured neurons to glutamate receptor agonist NMDA. Within 4 h, this agent caused a prolonged decrease in the activity of the ubiquitin-proteasome system as shown by disassembly of 26S proteasomes, decrease in ubiquitin-protein conjugates, and dissociation of the ubiquitin ligases UBE3A (E6-AP) and HUWE1 from the proteasome. Surprisingly, the regulatory 19S particles were rapidly degraded by proteasomal, not lysosomal degradation, and the dissociated E3 enzymes also degraded. Thus the content of proteasomes and their set of associated proteins can be altered by neuronal activity, in a manner likely to influence synaptic plasticity and learning.
 
Calretinin (CR) and calbindin D-28k (CB) are cytosolic EF-hand Ca2+-binding proteins and function as Ca2+ buffers affecting the spatiotemporal aspects of Ca2+ transients and possibly also as Ca2+ sensors modulating signaling cascades. In the adult hippocampal circuitry, CR and CB are expressed in specific principal neurons and subsets of interneurons. In addition, CR is transiently expressed within the neurogenic dentate gyrus (DG) niche. CR and CB expression during adult neurogenesis mark critical transition stages, onset of differentiation for CR and the switch to adult-like connectivity for CB. Absence of either protein during these stages in null-mutant mice may have functional consequences and contribute to some aspects of the identified phenotypes. We report the impact of CR- and CB-deficiency on the proliferation and differentiation of progenitor cells within the subgranular zone (SGZ) neurogenic niche of the DG. Effects were evaluated I) 2 and 4 weeks postnatally, during the transition period of the proliferative matrix to the adult state, and II) in adult animals (3 months) to trace possible permanent changes in adult neurogenesis. The absence of CB from differentiated DG granule cells has no retrograde effect on the proliferative activity of progenitor cells, nor affects survival or migration/differentiation of newborn neurons in the adult DG including the SGZ. On the contrary, lack of CR from immature early postmitotic granule cells causes an early loss in proliferative capacity of the SGZ that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells. The transient CR expression at the onset of adult neurogenesis differentiation may thus have two functions: I) to serve as a self-maintenance signal for the pool of cells at the same stage of neurogenesis contributing to their survival/differentiation, and II) it may contribute to retrograde signaling required for maintenance of the progenitor pool.
 
Effects of Mg2+ on Ca2+ binding. Simulated steady-state Ca2+-binding curves of the Ca2+ binding proteins (CaBP) parvalbumin (PV, red, KD,Ca 9 nM, KD,Mg 31 μM), calbindin (CB, black, average KD,Ca 393 nM, KD,Mg 714 μM; cf. Table 1), and calretinin (CR, green, average KD,Ca 1.5 μM, KD,Mg 4.5 mM) in the absence of Mg2+ (dotted lines) and in the presence of 600 μM Mg2+ (solid lines).
| Properties of calbindin D-28k.
Many neurons of the vertebrate central nervous system (CNS) express the Ca(2+) binding protein calbindin D-28k (CB), including important projection neurons like cerebellar Purkinje cells but also neocortical interneurons. CB has moderate cytoplasmic mobility and comprises at least four EF-hands that function in Ca(2+) binding with rapid to intermediate kinetics and affinity. Classically it was viewed as a pure Ca(2+) buffer important for neuronal survival. This view was extended by showing that CB is a critical determinant in the control of synaptic Ca(2+) dynamics, presumably with strong impact on plasticity and information processing. Already 30 years ago, in vitro studies suggested that CB could have an additional Ca(2+) sensor function, like its prominent acquaintance calmodulin (CaM). More recent work substantiated this hypothesis, revealing direct CB interactions with several target proteins. Different from a classical sensor, however, CB appears to interact with its targets both, in its Ca(2+)-loaded and Ca(2+)-free forms. Finally, CB has been shown to be involved in buffered transport of Ca(2+), in neurons but also in kidney. Thus, CB serves a threefold function as buffer, transporter and likely as a non-canonical sensor.
 
Schematic overview of the intricate regulation of PP2A enzymes. Major PP2A holoenzymes of this very large family (>96 enzymes) are heterotrimers containing a scaffolding “A” (one of two isoforms), a catalytic “C” (one of two isoforms), and one variable regulatory “B” subunit (one of twenty three isoforms). PP2A subunits are subjected to post-translational modifications, including methylation of the catalytic subunit on a conserved Leucine-309 residue, and phosphorylation. Endogenous subunit interactions, interaction of PP2A subunits with a variety of viral and cellular proteins, and binding of specific PP2A inhibitors and modulatory proteins to the catalytic subunit, all combine to modulate PP2A catalytic activity and ensure PP2A isoform-specific targeting and substrate specificity. Specific modulatory proteins also critically regulate PP2A biogenesis and stability. In addition, many compounds are known to enhance PP2A catalytic activity. See text for details.
Overview of PP2A dysfunction in AD and its link with the deregulation of tau. (A) Altered PP2A subunit expression, activity and post-translational modifications have been described in AD autopsy brain tissue. Some of these changes may be mediated by alterations in specific PP2A modulatory proteins (LCMT1, PTPA, alpha4) and endogenous PP2A inhibitors (I1PP2A and I2PP2A) that have also been reported in AD autopsy brain tissue. They also decrease the interaction of PP2A with tau. (B) The biogenesis of the PP2A/Bα holoenzyme, the primary Ser/Thr tau phosphatase in vivo, is believed to be controlled by Leu-309 methylation of PP2A catalytic subunit by LCMT1. This reaction requires the supply of SAM, the universal methyl donor, and is inhibited by SAH. The PP2A methylesterase, PME-1, can demethylate and inactivate PP2A through distinct mechanisms, and form a complex with inactive PP2A enzymes. Those inactive complexes could be-reactivated via the action of the PP2A activator PTPA, allowing for subsequent methylation of PP2A C subunit. Many brain Ser/Thr protein kinases, including GSK3β, oppose the action of PP2A/Bα and promote tau phosphorylation. Inhibition and/or down-regulation of PP2A can enhance tau phosphorylation directly by preventing its dephosphorylation, or indirectly by up-regulating tau kinases.
Deregulation of PP2A-tau protein–protein interactions in AD. (A) The PP2A/Bα holoenzyme can directly interact with three- or four-repeat human tau isoforms via a domain encompassing the microtubule-binding repeats (orange), resulting in tau dephosphorylation. A specific proline-rich motif (yellow) that contains the Thr231 phosphorylation site plays a critical role in modulating PP2A-tau protein–protein interactions. (B) PP2A-tau protein–protein interaction can be inhibited in vitro by: (1) Alterations in tau, including AD-like phosphorylation and FTDP-17 missense mutations; (2) decreased expression levels of PP2A methylation and PP2A/Bα in AD; (3) Fyn kinase and pseudophosphorylated RpTPPKSP peptides. Disruption of normal PP2A-tau interactions is predicted to affect tau phosphorylation state and function.
Model for the link between alterations in one-carbon metabolism, deregulation of PP2A methylation and AD-like pathology. Dietary B-vitamin deficiency, genetic polymorphisms in key enzymes that metabolize folate and homocysteine, drugs (e.g., the anti-folate drug, methotrexate), diseases (e.g., liver disease) and aging can all lead to impairment of one-carbon metabolism. In turn, alterations in the methylation cycle result in decreased LCMT1 activity and/or expression levels, and subsequent down-regulation of PP2A methylation and PP2A/Bα holoenzymes. This is associated with the accumulation of phosphorylated tau and APP proteins in vivo. Experiments in cultured cells and in vitro also link alterations in neuronal PP2A methylation with disruption of PP2A-tau protein–protein interactions, alterations in the subcellular targeting of PP2A and tau, APP processing, microtubule stability, and neuritic defects.
Protein phosphatase 2A (PP2A) is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer's disease (AD) field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. "PP2A" dysfunction has been linked to tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/Bα holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/Bα isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/Bα-tau protein interactions likely contribute to tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/Bα, enhanced phosphorylation of tau and amyloid precursor protein, tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie "PP2A" dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.
 
Serotonin (5-HT) appears to play a major role in controlling adult hippocampal neurogenesis and thereby it is relevant for theories linking failing adult neurogenesis to the pathogenesis of major depression and the mechanisms of action of antidepressants. Serotonergic drugs lacked acute effects on adult neurogenesis in many studies, which suggested a surprisingly long latency phase. Here we report that the selective serotonin reuptake inhibitor fluoxetine, which has no acute effect on precursor cell proliferation, causes the well-described increase in net neurogenesis upon prolonged treatment partly by promoting the survival and maturation of new postmitotic neurons. We hypothesized that this result is the cumulative effect of several 5-HT-dependent events in the course of adult neurogenesis. Thus, we used specific agonists and antagonists to 5-HT1a, 2, and 2c receptor subtypes to analyze their impact on different developmental stages. We found that 5-HT exerts acute and opposing effects on proliferation and survival or differentiation of precursor cells by activating the diverse receptor subtypes on different stages within the neuronal lineage in vivo. This was confirmed in vitro by demonstrating that 5-HT1a receptors are involved in self-renewal of precursor cells, whereas 5-HT2 receptors effect both proliferation and promote neuronal differentiation. We propose that under acute conditions 5-HT2 effects counteract the positive proliferative effect of 5-HT1a receptor activation. However, prolonged 5-HT2c receptor activation fosters an increase in late-stage progenitor cells and early postmitotic neurons, leading to a net increase in adult neurogenesis. Our data indicate that serotonin does not show effect latency in the adult dentate gyrus. Rather, the delayed response to serotonergic drugs with respect to endpoints downstream of the immediate receptor activity is largely due to the initially antagonistic and un-balanced action of different 5-HT receptors.
 
Circulating microRNAs, present either in the cellular component, peripheral blood mononuclear cells (PBMC), or in cell-free plasma, have emerged as biomarkers for age-dependent systemic, disease-associated changes in many organs. Previously, we have shown that microRNA (miR)-34a is increased in circulating PBMC of Alzheimer's disease (AD) patients. In the present study, we show that this microRNA's sister, miR-34c, exhibits even greater increase in both cellular and plasma components of AD circulating blood samples, compared to normal age-matched controls. Statistical analysis shows the accuracy of levels of miR-34c assayed by receiver operating characteristic (ROC) analysis: the area under the curve is 0.99 (p < 0.0001) and the 95% confidence level extends from 0.97 to 1. Pearson correlation between miR-34c levels and mild and moderate AD, as defined by the mini-mental state examination (MMSE), shows an r-value of -0.7, suggesting a relatively strong inverse relationship between the two parameters. These data show that plasma levels of microRNA 34c are much more prominent in AD than those of its sister, miR-34a, or than its own level in PBMC. Transfection studies show that miR-34c, as does its sister miR-34a, represses the expression of several selected genes involved in cell survival and oxidative defense pathways, such as Bcl2, SIRT1, and others, in cultured cells. Taken together, our results indicate that increased levels of miR-34c in both PBMC and plasma may reflect changes in circulating blood samples in AD patients, compared to age-matched normal controls.
 
MicroRNAs (miRNAs) are small, non-coding RNAs that function as key post-transcriptional regulators in neural development, brain function, and neurological diseases. Growing evidence indicates that miRNAs are also important mediators of nerve regeneration, however, the affected signaling mechanisms are not clearly understood. In the present study, we show that nerve injury-induced miR-431 stimulates regenerative axon growth by silencing Kremen1, an antagonist of Wnt/beta-catenin signaling. Both the gain-of-function of miR-431 and knockdown of Kremen1 significantly enhance axon outgrowth in murine dorsal root ganglion neuronal cultures. Using cross-linking with AGO-2 immunoprecipitation, and 3'-untranslated region (UTR) luciferase reporter assay we demonstrate miR-431 direct interaction on the 3'-UTR of Kremen1 mRNA. Together, our results identify miR-431 as an important regulator of axonal regeneration and a promising therapeutic target.
 
| Features of various vector systems.
Understanding how the CNS functions poses one of the greatest challenges in modern life science and medicine. Studying the brain is especially challenging because of its complexity, the heterogeneity of its cellular composition, and the substantial changes it undergoes throughout its life-span. The complexity of adult brain neural networks results also from the diversity of properties and functions of neuronal cells, governed, inter alia, by temporally and spatially differential expression of proteins in mammalian brain cell populations. Hence, research into the biology of CNS activity and its implications to human and animal behavior must use novel scientific tools. One source of such tools is the field of molecular genetics-recently utilized more and more frequently in neuroscience research. Transgenic approaches in general, and gene targeting in rodents have become fundamental tools for elucidating gene function in the CNS. Although spectacular progress has been achieved over recent decades by using these approaches, it is important to note that they face a number of restrictions. One of the main challenges is presented by the temporal and spatial regulation of introduced genetic manipulations. Viral vectors provide an alternative approach to temporally regulated, localized delivery of genetic modifications into neurons. In this review we describe available technologies for gene transfer into the adult mammalian CNS that use both viral and non-viral tools. We discuss viral vectors frequently used in neuroscience, with emphasis on lentiviral vector (LV) systems. We consider adverse effects of LVs, and the use of LVs for temporally and spatially controllable manipulations. Especially, we highlight the significance of viral vector-mediated genetic manipulations in studying learning and memory processes, and how they may be effectively used to separate out the various phases of learning: acquisition, consolidation, retrieval, and maintenance.
 
Acetylcholinesterase (AChE; EC 3.1.1.7) plays a crucial role in the rapid hydrolysis of the neurotransmitter acetylcholine, in the central and peripheral nervous system and might also participate in non-cholinergic mechanism related to neurodegenerative diseases. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-β (Aβ) peptide accumulation and synaptic alterations. We have previously shown that AChE is able to accelerate the Aβ peptide assembly into Alzheimer-type aggregates increasing its neurotoxicity. Furthermore, AChE activity is altered in brain and blood of Alzheimer's patients. The enzyme associated to amyloid plaques changes its enzymatic and pharmacological properties, as well as, increases its resistant to low pH, inhibitors and excess of substrate. Here, we reviewed the effects of IDN 5706, a hyperforin derivative that has potential preventive effects on the development of AD. Our results show that treatment with IDN 5706 for 10 weeks increases brain AChE activity in 7-month-old double transgenic mice (APP(SWE)-PS1) and decreases the content of AChE associated with different types of amyloid plaques in this Alzheimer's model. We concluded that early treatment with IDN 5706 decreases AChE-Aβ interaction and this effect might be of therapeutic interest in the treatment of AD.
 
Balancing excitation and inhibition. The majority of excitatory neurotransmitter receptors are presented on bulbous structures known as spines whereas inhibitory neurotransmitter receptors are usually present on postsynaptic sites formed on the dendritic shaft. (A) Homeostatic mechanisms maintain balance. In this example of synaptic scaling an increase in synaptic input results in a decrease in the number of neurotransmitter receptors present at particular synapses to balance changes in synaptic strength. (B) Scaffolding molecules regulate retention of cell adhesion molecules at excitatory vs. inhibitory contacts. Scaffolding molecules retain specific adhesion molecules at either excitatory or inhibitory synapses. An example illustrating a loss of a scaffolding molecule that controls retention of adhesion molecules at an inhibitory synapses may cause a reduction in the number of adhesion molecules retained at this site, and a corresponding shift of these adhesion molecules to excitatory synapses. This results in a shift in the E/I balance towards enhanced excitation. (C) PSD-95 regulates retention of AMPA-type glutamate receptors at excitatory synapses. AMPA receptors directly associate with stargazin molecules, which in turn associate with PSD-95. Reduction in PSD-95 levels at the synapse reduces AMPA receptor retention. This results in a shift in the E/I balance towards decreased excitation, or increased inhibition.
Macromolecular PSD-95 complex. The molecular organization of glutamatergic synapses is presented, but only major molecules associated with PSD-95 are shown. The various molecules portrayed regulate synapse function, morphology, trafficking and localization of adhesion molecules and neurotransmitter receptors.
Excitability of individual neurons dictates the overall excitation in specific brain circuits. This process is thought to be regulated by molecules that regulate synapse number, morphology and strength. Neuronal excitation is also influenced by the amounts of neurotransmitter receptors and signaling molecules retained at particular synaptic sites. Recent studies revealed a key role for PSD-95, a scaffolding molecule enriched at glutamatergic synapses, in modulation of clustering of several neurotransmitter receptors, adhesion molecules, ion channels, cytoskeletal elements and signaling molecules at postsynaptic sites. In this review we will highlight mechanisms that control targeting of PSD-95 at the synapse, and discuss how this molecule influences the retention and clustering of diverse synaptic proteins to regulate synaptic structure and strength. We will also discuss how PSD-95 may maintain a balance between excitation and inhibition in the brain and how alterations in this balance may contribute to neuropsychiatric disorders.
 
Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.
 
Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca(2+)-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM-the A(2A) adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A(2A)-NCS-1 complexes in living cells whereas caldendrin did not associate with A(2A) receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A(2A) receptor intracellular signaling in a Ca(2+)-dependent manner. Taken together this study provides further evidence that neuronal Ca(2+)-sensor proteins play an important role in modulation of GPCR signaling.
 
Gene therapies for neurological diseases with autonomic or gastrointestinal involvement may require global gene expression. Gastrointestinal complications are often associated with Parkinson's disease and autism. Lewy bodies, a pathological hallmark of Parkinson's brains, are routinely identified in the neurons of the enteric nervous system (ENS) following colon biopsies from patients. The ENS is the intrinsic nervous system of the gut, and is responsible for coordinating the secretory and motor functions of the gastrointestinal tract. ENS dysfunction can cause severe patient discomfort, malnourishment, or even death as in intestinal pseudo-obstruction (Ogilvie syndrome). Importantly, ENS transduction following systemic vector administration has not been thoroughly evaluated. Here we show that systemic injection of AAV9 into neonate or juvenile mice results in transduction of 25-57% of ENS myenteric neurons. Transgene expression was prominent in choline acetyltransferase positive cells, but not within vasoactive intestinal peptide or neuronal nitric oxide synthase cells, suggesting a bias for cells involved in excitatory signaling. AAV9 transduction in enteric glia is very low compared to CNS astrocytes. Enteric glial transduction was enhanced by using a glial specific promoter. Furthermore, we show that AAV8 results in comparable transduction in neonatal mice to AAV9 though AAV1, 5, and 6 are less efficient. These data demonstrate that systemic AAV9 has high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies of the ENS.
 
| Stimulants of mTOR in CNS neurons.
Upper panel. Domain structure of mTOR. HEAT: Huntington Elongation Factor 3 PR65/A TOR, FAT: FRAP ATM TTRAP, FRB: FKBP12-Rapamycin Binding. Middle and lower panel: Components of mTOR complexs.
The flow sheet of upstream and downstream of mTORCs. Representative substrates and cellular responses mentioned in the text are shown. Note that mTORC2 activates mTORC1 through Akt.
Scheme of translation processes that are regulated by mTORC1. Upper panel: translation initiation. mTORC1 directly phosphorylates 4EBP and liberates eIF4E. eIF4E with mRNA then binds to eIF4G to form eIF4F complex. Phosphorylation of eIF4G and eIF4B is mTORC1-dependent. Assembly of eIF3 subunits and eIF4G is also thought to be mTORC1-dependent. Lower panel: translation elongation. p70S6K downstream of mTORC1 phosphorylates eEF2K and suppresses its activity to phosphorylate eEF2. Non-phosphorylated form of eEF2 is an active form thus enhances elongation process.
A graphic of hypothetical neuronal development governed by mTORC1. Neurons receive nutrients globally and growth factors/transmitters locally. Both inputs coordinately activate mTORC1 that leads normal neuronal development. Suppression or overactivation of mTORC1 result dysregulation of neuronal morphology and function. (note that photographs of a neuron was image processed).
Target of rapamycin (TOR) was first identified in yeast as a target molecule of rapamycin, an anti-fugal and immunosuppressant macrolide compound. In mammals, its orthologue is called mammalian TOR (mTOR). mTOR is a serine/threonine kinase that converges different extracellular stimuli, such as nutrients and growth factors, and diverges into several biochemical reactions, including translation, autophagy, transcription, and lipid synthesis among others. These biochemical reactions govern cell growth and cause cells to attain an anabolic state. Thus, the disruption of mTOR signaling is implicated in a wide array of diseases such as cancer, diabetes, and obesity. In the central nervous system, the mTOR signaling cascade is activated by nutrients, neurotrophic factors, and neurotransmitters that enhances protein (and possibly lipid) synthesis and suppresses autophagy. These processes contribute to normal neuronal growth by promoting their differentiation, neurite elongation and branching, and synaptic formation during development. Therefore, disruption of mTOR signaling may cause neuronal degeneration and abnormal neural development. While reduced mTOR signaling is associated with neurodegeneration, excess activation of mTOR signaling causes abnormal development of neurons and glia, leading to brain malformation. In this review, we first introduce the current state of molecular knowledge of mTOR complexes and signaling in general. We then describe mTOR activation in neurons, which leads to translational enhancement, and finally discuss the link between mTOR and normal/abnormal neuronal growth during development.
 
The Ubiquitin Proteasome System and its components regulated after drug exposure. (A) Schematic representation of the Ubiquitin Proteasome System. The external and internal rings constitute the 20S proteasome. The lid and base constitute the 19S regulatory complex. In some cases, it can be replaced by the PA28 or 11S regulatory complex, constituted of a single ring of 7 subunits. (B) Classification of the UPS components found to be regulated after drug exposure.
UPS involvement in behavioral sensitization and reconsolidation of morphine place preference. (A) Schematic representation of the protocol followed in context-dependent and -independent locomotor sensitization. The morphine dose was 10 mg/kg. (B) UPS inhibition blocks the acquisition of behavioral sensitization when a context-dependent paradigm is used (lactacystin 100 pmol in 0.5 μl per side: n = 8 and DMSO: n = 11), (C) whereas it does not affect this drug-adaptation in a context-independent procedure (lactacystin: n = 6 and DMSO: n = 8). Data are expressed in number of beam breaks ± SEM during a 1 h session after morphine injection before (day 1; empty bars) and after conditioning (day 8; black bars). Two-Way ANOVA followed by Bonferroni post-hoc tests: n.s., non-significant; ***p < 0.001. (D) Schematic representation of the protocol used for assessing the role of the UPS in reconsolidation. (E) Intra-Nacc bilateral injection of lactacystin 1 h before a drug-context re-exposure abolishes drug-induced place preference when tested 24 h after this new association (n = 6) whereas DMSO treated-animals still express place preference (n = 6). Data are expressed as percentage of time spent in the drug-associated compartment ± SEM during pre-conditioning tests (empty bars) and post-conditioning tests (filled bars). Two-Way ANOVA followed by Bonferroni post-hoc tests: n.s., non-significant; *p < 0.05. See Massaly et al. (2013) for details about behavioral procedures.
Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors.
 
Alcohol affects local translation of proteins in amygdala of alcohol-dependent rats. Alcohol exposure using conditioning chambers, as well as a non-pharmacologically active alcohol injection (priming) that served as an odor-taste cue, increased mTORC1 (mammalian target of rapamycin complex 1) activation and the phosphorylation/activation of its downstream substrates, such as eukaryotic translation initiation factor-4E binding protein (4E-BP), S6 kinase (S6K) and the S6K substrates, and increased expression of post-synaptic density-95 (PSD95) and the AMPA receptor GluA1 subunit (Barak et al., 2013). These effects were abolished by the mTORC1 inhibitor, rapamycin. A second pathway by which alcohol mediates synaptic translation is by increasing extracellular glutamate. This leads to activation of voltage-dependent Ca2+ channels (VDCCs) and activity-dependent release of brain-derived neurotrophic factor (BDNF). BDNF then binds and stimulates its receptor, tropomyosin-related kinase B (TrkB), activating downstream signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), Akt and Ras, stimulating mTORC1, which culminates in the signaling effects described above. The increased GluA1-subunits are inserted into the membrane, increasing excitation and alcohol-sensitive substrates. Thus, mTORC1 activation mediates the translation of specific synaptic proteins that are important in plasticity processes. Figure and legend were adapted from Duman and Voleti (2012). Abbreviations: 4E-BP, 4E binding protein; eEF2K, eukaryotic elongation factor-2 kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; P indicates phosphorylated protein.
Model for cocaine-induced microRNA-mRNA interactions. Cocaine causes the down-regulation of let-7d, resulting in induction of its target genes, BDNF and cAMP-responsive element-binding protein (CREB). In contrast, miR-181a is up-regulated by cocaine, causing down-regulation of its targets, GRM5 (glutamate metabotropic receptor 5) and AMPA receptor subunit 2 (GluA2). These data support the involvement of let-7d and miR-181a in regulating the differential expression of various genes in response to cocaine, which may impact molecular adaptations leading to addiction. Figure and legend were modified from the original in Jonkman and Kenny (2013).
The interactions between miR-212, CREB, methyl CpG-binding protein 2 (MeCP2), and BDNF. Cocaine activates CREB-miR-212 and MeCP2-BDNF signaling and the balance between these pathways likely regulates escalation of cocaine intake (‘loss of control’). The orange circle illustrates CREB signaling, which protects against the development of escalating cocaine intake, whereas the green circle shows that MeCP2-BDNF signaling promotes escalation of intake. Green arrows indicate a stimulatory relationship, whereas red lines indicate an inhibitory relationship. Figure and legend were taken from Jonkman and Kenny (2013).
Local translation of mRNAs is a mechanism by which cells can rapidly remodel synaptic structure and function. There is ample evidence for a role of synaptic translation in the neuroadaptations resulting from chronic drug use and abuse. Persistent and coordinated changes of many mRNAs, globally and locally, may have a causal role in complex disorders such as addiction. In this review we examine the evidence that translational regulation by microRNAs drives synaptic remodeling and mRNA expression, which may regulate the transition from recreational to compulsive drug use. microRNAs are small, non-coding RNAs that control the translation of mRNAs in the cell and within spatially restricted sites such as the synapse. microRNAs typically repress the translation of mRNAs into protein by binding to the 3'UTR of their targets. As 'master regulators' of many mRNAs, changes in microRNAs could account for the systemic alterations in mRNA and protein expression observed with drug abuse and dependence. Recent studies indicate that manipulation of microRNAs affects addiction-related behaviors such as the rewarding properties of cocaine, cocaine-seeking behavior, and self-administration rates of alcohol. There is limited evidence, however, regarding how synaptic microRNAs control local mRNA translation during chronic drug exposure and how this contributes to the development of dependence. Here, we discuss research supporting microRNA regulation of local mRNA translation and how drugs of abuse may target this process. The ability of synaptic microRNAs to rapidly regulate mRNAs provides a discrete, localized system that could potentially be used as diagnostic and treatment tools for alcohol and other addiction disorders.
 
Changes in AMPAR-mediated synaptic transmission rectification index (RI) measured in NAc core MSNs following withdrawal from extended access cocaine self-administration. Compared to saline-treated animals (n = 17, pooled from withdrawal days [WD] 35–70), no apparent changes in RI were observed during the first month of withdrawal from cocaine self-administration (WD16, n = 5; WD25, n = 5). However, a significant increase in RI was observed after a month of withdrawal (WD30–35, n = 7). The increased RI persisted through WD70 (WD35–45, n = 10; WD45–55, n = 13; WD55–70, n = 7), the latest time-point measured. *P < 0.01 vs. saline, Tukey post-hoc test after significant ANOVA.
| Summary of AMPAR changes in VTA and NAc after different cocaine regimens.
| Continued.
In animal models of drug addiction, cocaine exposure has been shown to increase levels of calcium-permeable AMPA receptors (CP-AMPARs) in two brain regions that are critical for motivation and reward - the ventral tegmental area (VTA) and the nucleus accumbens (NAc). This review compares CP-AMPAR plasticity in the two brain regions and addresses its functional significance. In VTA dopamine neurons, cocaine exposure results in synaptic insertion of high conductance CP-AMPARs in exchange for lower conductance calcium-impermeable AMPARs (CI-AMPARs). This plasticity is rapid (hours), GluA2-dependent, and can be observed with a single cocaine injection. In addition to strengthening synapses and altering Ca2+ signaling, CP-AMPAR insertion affects subsequent induction of plasticity at VTA synapses. However, CP-AMPAR insertion is unlikely to mediate the increased dopamine cell activity that occurs during early withdrawal from cocaine exposure. Within the VTA, the group I metabotropic glutamate receptor mGluR1 exerts a negative influence on CP-AMPAR accumulation. Acutely, mGluR1 stimulation elicits a form of LTD resulting from CP-AMPAR removal and CI-AMPAR insertion. In medium spiny neurons (MSNs) of the NAc, extended access cocaine self-administration is required to increase CP-AMPAR levels. This is first detected after approximately a month of withdrawal and then persists. Once present in NAc synapses, CP-AMPARs mediate the expression of incubation of cue-induced cocaine craving. The mechanism of their accumulation may be GluA1-dependent, which differs from that observed in the VTA. However, similar to VTA, mGluR1 stimulation removes CP-AMPARs from MSN synapses. Loss of mGluR1 tone during cocaine withdrawal may contribute to CP-AMPAR accumulation in the NAc. Thus, results in both brain regions point to the possibility of using positive modulators of mGluR1 as a treatment for cocaine addiction.
 
| Optogenetic activation of NAc D1-MSNs but not D2-MSNs alters Tiam1 levels. (A) D1-Cre or D2-Cre mice expressing AAV-DIO-ChR2-EYFP or AAV-DIO-EYFP in NAc received 5 days of blue light illumination to the NAc (n = 4 per group) and NAc tissue was used to perform gene expression analyzes with the Mouse Cytoskeleton Regulators PCR array (SA Biosciences). We find that three genes were significantly regulated in D1-ChR2 NAc compared to D1-EYFP NAc, four genes were significantly regulated in D2-ChR2 NAc compared to D2-EYFP, and three genes were significantly regulated in both D1-ChR2 and D2-ChR2 compared to EYFP controls. Tiam1 was a gene that was significantly down-regulated by repeated D1-ChR2 activation but not by D2-ChR2 activation. Heat maps are expressed
Tiam1 is decreased in the NAc of rats that self-administer cocaine.(A) Self-administration of cocaine (1.0 mg/kg/infusion, FR1 schedule) or saline over a 10-day period. Rats self-administering cocaine took more infusions than saline controls on days 2–10 of testing (n = 6 per group, two-way repeated measures ANOVA, F(9,90) = 4.139, p < 0.0001, post hoc test, p < 0.05). (B) Following cocaine self-administration, Tiam1 mRNA is significantly decreased in the NAc compared to saline controls (n = 6 per group, Student”s t-test, **p < 0.01). (C) Tiam1 protein levels are similarly down-regulated in the NAc in rats that self-administer cocaine compared to saline controls (n = 4–7 per group, Student”s t-test, **p < 0.01).
Optogenetic activation of NAc D1-MSNs but not D2-MSNs alters Tiam1 levels. (A) D1-Cre or D2-Cre mice expressing AAV-DIO-ChR2-EYFP or AAV-DIO-EYFP in NAc received 5 days of blue light illumination to the NAc (n = 4 per group) and NAc tissue was used to perform gene expression analyzes with the Mouse Cytoskeleton Regulators PCR array (SA Biosciences). We find that three genes were significantly regulated in D1-ChR2 NAc compared to D1-EYFP NAc, four genes were significantly regulated in D2-ChR2 NAc compared to D2-EYFP, and three genes were significantly regulated in both D1-ChR2 and D2-ChR2 compared to EYFP controls. Tiam1 was a gene that was significantly down-regulated by repeated D1-ChR2 activation but not by D2-ChR2 activation. Heat maps are expressed in log scale. (B) D2-Cre mice expressing AAV-DIO-ChR2-EYFP or AAV-DIO-EYFP in NAc were injected (i.p.) with saline (day 0) followed by injections (i.p.) with either cocaine (10 mg/kg) or saline coupled with blue light illumination to the NAc and locomotor activity was monitored for 1 h (days 1–5, n = 5–6 per group). D2-MSN activation in the AAV-DIO-ChR2-EYFP cocaine group resulted in a significant attenuation in cocaine locomotor activity on days 4 and 5 compared to the AAV-DIO-EYFP cocaine group. [Repeated measure two-way ANOVA, F(10, 70) = 7.46, p < 0.001, post hoc test, **p < 0.01.] (C) No difference is observed in Tiam1 mRNA levels in AAV-DIO-EYFP and AAV-DIO-ChR2-EYFP cocaine groups (n = 5–6).
Optogenetic inhibition of D1-MSNs.(A) Immunofluorescence image of D1-Cre NAc expressing eNpHR3.0-EYFP (green) in Cre positive D1-MSNs (red; scale bar, 25 μm). (B) Hyperpolarization and (C)outward photo-current of the membrane potential in eNpHR3.0 expressing D1-MSNs during green (532 nm) light illumination. (D) Inhibition of neuronal firing during green (532 nm) light illumination in NAc D1-MSNs expressing eNpHR3.0. (E) 5-day, 60-min per day green light illumination (paradigm used in Figure 4) to the NAc does not alter cell death markers or NeuN gene expression in D1-Cre NAc expressing AAV-DIO-eNpHR3.0-EYFP or AAV-DIO-EYFP (Student”s t-test, p > 0.05).
Optogenetic inhibition of NAc D1-MSNs attenuates cocaine locomotor sensitization and reverses Tiam1 gene expression. (A) D1-Cre mice expressing AAV-DIO-eNpHR3.0-EYFP or AAV-DIO-EYFP in NAc were injected (i.p.) with saline (day 0) followed by injections (i.p.) with either cocaine (10 mg/kg) or saline coupled with green light illumination to the NAc and locomotor activity was monitored for 1 h (days 1–5, n = 4–7 per group). D1-MSN inhibition in the AAV-DIO-eNpHR3.0-EYFP cocaine group resulted in a significant attenuation in cocaine locomotor activity on days 2–4 compared to the AAV-DIO-EYFP cocaine group. (Repeated measure two-way ANOVA, F(15,85) = 13.03, p < 0.0001, post hoc test, *p < 0.05.) (B,C) Tiam1 mRNA and protein is significantly down-regulated in the NAc of the cocaine AAV-DIO-EYFP group compared to the saline AAV-DIO-EYFP and AAV-DIO-eNpHR3.0-EYFP saline groups. Inhibition of D1-MSNs in the AAV-DIO-eNpHR3.0 cocaine group reversed the blunted Tiam1 cocaine response (mRNA: n = 4–7 per group, two-way ANOVA, interaction, F(1,16) = 7.64, p < 0.05, post hoc test, *p < 0.05; protein: n = 4–7 per group, two-way ANOVA, group effect, F(1,16) = 6.24, p < 0.05, drug effect, F(1,16) = 4.60, p < 0.05, post hoc test, *p < 0.05).
Exposure to psychostimulants results in structural and synaptic plasticity in striatal medium spiny neurons (MSNs). These cellular adaptations arise from alterations in genes that are highly implicated in the rearrangement of the actin-cytoskeleton, such as T-lymphoma invasion and metastasis 1 (Tiam1). Previous studies have demonstrated a crucial role for dopamine receptor 1 (D1)-containing striatal MSNs in mediating psychostimulant induced plasticity changes. These D1-MSNs in the nucleus accumbens (NAc) positively regulate drug seeking, reward, and locomotor behavioral effects as well as the morphological adaptations of psychostimulant drugs. Here, we demonstrate that rats that actively self-administer cocaine display reduced levels of Tiam1 in the NAc. To further examine the cell type-specific contribution to these changes in Tiam1 we used optogenetics to selectively manipulate NAc D1-MSNs or dopamine receptor 2 (D2) expressing MSNs. We find that repeated channelrhodopsin-2 activation of D1-MSNs but not D2-MSNs caused a down-regulation of Tiam1 levels similar to the effects of cocaine. Further, activation of D2-MSNs, which caused a late blunted cocaine-mediated locomotor behavioral response, did not alter Tiam1 levels. We then examined the contribution of D1-MSNs to the cocaine-mediated decrease of Tiam1. Using the light activated chloride pump, eNpHR3.0 (enhanced Natronomonas pharaonis halorhodopsin 3.0), we selectively inhibited D1-MSNs during cocaine exposure, which resulted in a behavioral blockade of cocaine-induced locomotor sensitization. Moreover, inhibiting these NAc D1-MSNs during cocaine exposure reversed the down-regulation of Tiam1 gene expression and protein levels. These data demonstrate that altering activity in specific neural circuits with optogenetics can impact the underlying molecular substrates of psychostimulant-mediated behavior and function.
 
Brain cells expend large amounts of energy sequestering calcium (Ca(2+)), while loss of Ca(2+) compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P), a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum (ER) to sequester Ca(2+). This led to the hypothesis that G6P regulates Ca(2+) accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA). Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, (45)Ca(2+) accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi) coupled with Ca(2+) accumulation was quantified. Addition of G6P significantly and decreased Ca(2+) accumulation in a dose-dependent fashion (1-10 mM). The reduction in Ca(2+) accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca(2+) accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca(2+) uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca(2+) dystasis caused by altered G6P regulation of SERCA activity.
 
Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in different brain regions that include the ventral tegmental area (VTA), nucleus accumbens (NAc), hippocampus, prefrontal cortex (PFC), and amygdala. Activation of nAChRs in these brain areas significantly contribute to the rewarding effects of ethanol and nicotine and play a role in modulating synaptic plasticity. GABAergic (red), glutamatergic (green), and dopaminergic (blue) connections between these structures constitute a major neural circuitry underlying addictive disorders.
Schematic representation of nAChR subtypes and circuit function in the mesolimbic dopaminergic system. (A) Pyramidal cells in layer V of the PFC lack nAChRs but their activity is modulated by excitatory and inhibitory neurons that do express them. There are two types of GABAergic interneurons, fast spiking and non-fast spiking, with only the latter bearing nAChRs (α7 and α4β2*). Distinct populations of glutamatergic inputs express either α7 or α4β2* nAChRs while DA terminals projecting from the VTA contain α4β2* nAChRs. Cholinergic inputs into the PFC arise from the nucleus basalis of Meynert (nBM). (B) In the NAc, nAChRs (α4β2*, α6β2*, and α6α4β2*) expressed on DAergic terminals from the VTA mediate DA release based on the neuronal activity firing rate. A small population of tonically active cholinergic interneurons (~2%) is synchronized with DA cell firing. Glutamatergic inputs from the PFC endow α7 nAChRs. (C) The VTA receives cholinergic innervation from the pedunculopontine (PPn) and laterodorsal tegmental nuclei (LDTn). In addition to the nAChRs localized on DA cell bodies, DAergic cell firing is modulated by α4β2* (and possibly α7) nAChRs expressed on GABAergic interneurons and excitatory glutamatergic afferents from the PFC and the PPn.
| nAChRs modulate synaptic plasticity in the hippocampus and amygdala.
Schematic representation of nAChR subtypes and circuit function in the hippocampus and amygdala. (A) In the hippocampus, α7 and α4β2* nAChRs are abundantly expressed on pyramidal cells and inhibitory interneurons. GABAergic interneurons have pre-synaptic α7 nAChRs and somato-dendritic expression of α7 and α4β2* nAChRs. Glutamatergic afferents have predominately pre-synaptic α7 nAChRs and only low levels of α3β4*. (B) In the amygdala, cholinergic inputs from the basal forebrain synapse in proximity to pre-synaptic nAChRs that modulate both excitatory and inhibitory synaptic transmission. Glutamatergic afferents and pyramidal neurons endow α7 nAChRs and GABAergic interneurons express multiple nAChRs (α7, α4β2*, and α3β4*).
Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug's reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine's effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies.
 
| Continued
Schematic overview of ACh synthesis, packaging, and destruction. (A) The choline acetyl transferase (ChAT) gene responsible for ACh synthesis is 56 kb in size and all its splice variants have the same 3′UTR. (B) The vesicular acetylcholine transporter (VAChT) controlling ACh packaging into vesicles is encoded from the first intron of the ChAT gene. The VAChT gene is only 2 kb in length, yet it has its own 3′UTR. Once released, ACh should be degraded into Acetate and Choline by one of the splice variants of AChE or BChE, depending on its site of release. (C,D) The AChE gene is 6 kb in size, and its mRNA transcript is spliced to yield the major AChE-S and AChE-R splice variants with distinct 3′-UTR domains. (E) The BChE gene is 65 kb in size and yields only one known splice variant.
MiRNAs targeting AChE-S and AChE-R, BChE and AChE-R or VAChT and AChE-R show associations to different disease groups related to cholinergic signaling. The pie chart classifies the shared targets, as well as the relevant diseases and highlights the observed interactions between them. Disease group association is shown as surrounding lines of differential thicknesses, reflecting the number of miRNAs in each group. The red, blue, green, orange and purple colored lines symbolize disease associations with the groups of “inflammation and anxiety,” “brain damage (e.g., stroke),” “cardiac diseases,” “neurodegenerative diseases,” and “pain,” respectively. Note that miR-132 and miR-125b associate with all disease groups. The monkey heads next to specific miRNA numbers symbolize primate specificity.
Intestinal miR-186 increases under predator scent stress are accompanied by elevated BChE and AChE activities. Top: The nucleotide sequence of the BChE and AChE-R-targeting miR-186 and schemes of its AChE-R and BChE mRNA targets. The “seed” sequence is underlined. (A) qRT-PCR quantification normalized to RNU6 levels demonstrates reproducible RFU values and 1.6-fold excess of miR-186 in intestinal sections from stressed mice, 7 days post-predator scent exposure (p = 0.016). (B,C) Highly significant intestinal elevation of total acetylthiocholine hydrolytic activity (p = 0.0032) accompanied by less pronounced AChE activity measured in the presence of 10 μM iso-OMPA (p = 0.05). N = 5 mice per group, in all tests.
Disease association of predicted CholinomiRs.
MicroRNAs (miRNAs) can notably control many targets each and regulate entire cellular pathways, but whether miRNAs can regulate complete neurotransmission processes is largely unknown. Here, we report that miRNAs with complementary sequence motifs to the key genes involved in acetylcholine (ACh) synthesis and/or packaging show massive overlap with those regulating ACh degradation. To address this topic, we first searched for miRNAs that could target the 3'-untranslated regions of the choline acetyltransferase (ChAT) gene that controls ACh synthesis; the vesicular ACh transporter (VAChT), encoded from an intron in the ChAT gene and the ACh hydrolyzing genes acetyl- and/or butyrylcholinesterase (AChE, BChE). Intriguingly, we found that many of the miRNAs targeting these genes are primate-specific, and that changes in their levels associate with inflammation, anxiety, brain damage, cardiac, neurodegenerative, or pain-related syndromes. To validate the in vivo relevance of this dual interaction, we selected the evolutionarily conserved miR-186, which targets both the stress-inducible soluble "readthrough" variant AChE-R and the major peripheral cholinesterase BChE. We exposed mice to predator scent stress and searched for potential associations between consequent changes in their miR-186, AChE-R, and BChE levels. Both intestinal miR-186 as well as BChE and AChE-R activities were conspicuously elevated 1 week post-exposure, highlighting the previously unknown involvement of miR-186 and BChE in psychological stress responses. Overlapping miRNA regulation emerges from our findings as a recently evolved surveillance mechanism over cholinergic neurotransmission in health and disease; and the corresponding miRNA details and disease relevance may serve as a useful resource for studying the molecular mechanisms underlying this surveillance.
 
A common feature in the Alzheimer's disease (AD) brain is the presence of acetylcholinesterase (AChE) which is commonly associated with β-amyloid plaques and neurofibrillary tangles (NFT). Although our understanding of the relationship between AChE and the pathological features of AD is incomplete, increasing evidence suggests that both β-amyloid protein (Aβ) and abnormally hyperphosphorylated tau (P-tau) can influence AChE expression. We also review recent findings which suggest the possible role of AChE in the development of a vicious cycle of Aβ and P-tau dysregulation and discuss the limited and temporary effect of therapeutic intervention with AChE inhibitors.
 
Two types of AChE–BChE hybrid enzyme. (A) The PRiMA-linked G4 AChE–BChE hybrid in chicken brain. (B) The ColQ-linked A12 AChE–BChE hybrid in chicken muscle.
Model for the assembly of PRiMA-linked AChE and BChE tetramers. When AChET and BChET subunits are expressed together, they form their own homodimers spontaneously. PRiMA recruits two homodimers together to form a PRiMA-linked tetramers, e.g., G4 AChE, G4 BChE. G4 hybrid.
Comparison of FHB sequences of AChET and BChET among different species. The sequences of the two alpha helices (FHB-1 and FHB-2) forming the dimeric contact zone of AChE and BChE are shown. The residues conserved across species are highlighted in bold. The amino acid sequences of human, mouse, rat, chicken, and Torpedo AChE catalytic subunits were deduced from nucleotide sequences accessed from GeneBank™ AAA68151, CAA39867, EDM13278, P36196, and CAA27169, respectively. The amino acid sequences of human, mouse, rat, and chicken BChE catalytic subunits were deduced from nucleotide sequences accessed from GeneBank™ AAA99296.1, AAH99977, NP_075231, and NP_989977, respectively.
Three types of interaction involved in the oligomerization of AChE and BChE. (A) The FHB domains of AChET or BChET direct the dimer formation. (B) The t-peptides of AChET or BChET form intercatenary disulfide bonds. (C) WAT domains of AChET and BChET interact with PRAD on PRiMA or ColQ.
Proposed model for the assembly and membrane processing of G4 ChEs. G4 AChE, G4 BChE, and AChE–BChE G4 hybrid molecules are assembled in ER where both AChET and BChET subunits have initial glycosylation. These G4 complexes are subsequently transported to Golgi apparatus where the catalytic subunits can have further glycosylation, and finally anchored onto the plasma membrane. The AChET glycosylation mutant, in which the glycosylation is completely abolished, is still able to assembly with PRiMA and BChET to form G4 AChE and G4 hybrid. However, both of them are retained in ER, which possibly will be subjected to the degradation pathway.
Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.
 
Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM) shRNA for AChE was used to inhibit AChE protein expression. Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the mRNA and protein level. Inhibition of AChE protein expression by shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R isoform, is involved in the apoptotic pathway caused by hyperglycemia in Y79 cells.
 
To date, more than 40 different types of cells from primary cultures or cell lines have shown AChE expression during apoptosis and after the induction apoptosis by different stimuli. It has been well-established that increased AChE expression or activity is detected in apoptotic cells after apoptotic stimuli in vitro and in vivo, and AChE could be therefore used as a marker of apoptosis. AChE is not an apoptosis initiator, but the cells in which AChE is overexpressed undergo apoptosis more easily than controls. Interestingly, cells with downregulated levels of AChE are not sensitive to apoptosis induction and AChE deficiency can protect against apoptosis. Some tumor cells do not express AChE, but when AChE is introduced into a tumor cell, the cells cease to proliferate and undergo apoptosis more readily. Therefore, AChE can be classified as a tumor suppressor gene. AChE plays a pivotal role in apoptosome formation, and silencing of the AChE gene prevents caspase-9 activation, with consequent decreased cell viability, nuclear condensation, and poly (adenosine diphosphate-ribose) polymerase cleavage. AChE is translocated into the nucleus, which may be an important event during apoptosis. Several questions still need to be addressed, and further studies that address the non-classical function of AChE in apoptosis are needed.
 
Hematopoietic stem cells (HSCs) differentiate and generate all blood cell lineages while maintaining self-renewal ability throughout life. Systemic responses to stressful insults, either psychological or physical exert both stimulating and down-regulating effects on these dynamic members of the immune system. Stress-facilitated division and re-oriented differentiation of progenitor cells modifies hematopoietic cell type composition, while enhancing cytokine production and promoting inflammation. Inversely, stress-induced increases in the neurotransmitter acetylcholine (ACh) act to mitigate inflammatory response and regain homeostasis. This signaling process is terminated when ACh is hydrolyzed by acetylcholinesterase (AChE). Alternative splicing, which is stress-modified, changes the composition of AChE variants, modifying their terminal sequences, susceptibility for microRNA suppression, and sub-cellular localizations. Intriguingly, the effects of stress and AChE variants on hematopoietic development and inflammation in health and disease are both subject to small molecule as well as oligonucleotide-mediated manipulations in vitro and in vivo. The therapeutic agents can thus be targeted to the enzyme protein, its encoding mRNA transcripts, or the regulator microRNA-132, opening new venues for therapeutic interference with multiple nervous and immune system diseases.
 
| A model illustrating post-transcriptional regulation of AChE expression and localization in neurons by trans-acting factors. (A) Alternative splicing of AChE pre-mRNA is controlled by SC35 and ASF/SF2 general splicing factors. As part of a ribonucleoprotein particle (RNP), nELAVs might also bind to cis-elements within the AChE pre-mRNA to regulate alternative splicing. (B) AChE mRNA is stabilized by HuD and possibly other nELAVs. Stabilization of AChE mRNA could
Schematic diagrams depicting the mouse and human AChE genes, alternative splicing options and cis-element locations. (A) Mouse and human AChE genes, including the leader exons identified in brain tissues. Noteworthy is the 5′ regulatory region that harbors alternate exons 1a–e in mouse and 1a–d in human (orange boxes), distal enhancer glucocorticoid response element (brown line) and proximal muscle-specific enhancer (pink line). Gray boxes and black lines represent constitutive exons and introns, respectively. (B) Alternative splicing at the 3′ end of AChE pre-mRNA produces tissue-specific R (green), H (yellow) and T (blue) variants. Approximate locations of the U-rich regulatory sequence, SC35 and ASF/SF2 splicing factor binding sites are denoted by black, white, and gray stars, respectively. (C) The 3′ untranslated regions (3′ UTRs) of mature T and R AChE variants and the alternative polyadenylation-dependent extended regions. The AChEH variant is not shown since it is not significantly expressed in neurons. Symbols represent the PBE (black triangle), ARE (gray triangle), miR-132 binding (white triangle), translation stop (red octagon), and polyadenylation signal (p(A)) sites. 4′ signifies a pseudo-intron that contains the AChER translation termination site. Whether E6 and the downstream region are included in AChER transcripts is unknown (?).
A model illustrating post-transcriptional regulation of AChE expression and localization in neurons by trans-acting factors. (A) Alternative splicing of AChE pre-mRNA is controlled by SC35 and ASF/SF2 general splicing factors. As part of a ribonucleoprotein particle (RNP), nELAVs might also bind to cis-elements within the AChE pre-mRNA to regulate alternative splicing. (B) AChE mRNA is stabilized by HuD and possibly other nELAVs. Stabilization of AChE mRNA could depend on nELAVs outcompeting destabilizing RBPs and/or RISC loaded miR-132, thereby preventing exosome-mediated mRNA degradation. (C) RNPs, conceivably containing nELAVs and Pumilio 2 (Pum2), transport translationally repressed AChE transcripts along microtubules into neurites. (D) At the synaptic terminal, AChE translation might be promoted by nELAVs or inhibited by Pum2 or RISC loaded miR-132.
The most characterized function of acetylcholinesterase (AChE) is to terminate cholinergic signaling at neuron-neuron and neuro-muscular synapses. In addition, AChE is causally or casually implicated in neuronal development, stress-response, cognition, and neurodegenerative diseases. Given the importance of AChE, many studies have focused on identifying the molecular mechanisms that govern its expression. Despite these efforts, post-transcriptional control of AChE mRNA expression is still relatively unclear. Here, we review the trans-acting factors and cis-acting elements that are known to control AChE pre-mRNA splicing, mature mRNA stability and translation. Moreover, since the Hu/ELAV family of RNA-binding proteins (RBPs) have emerged in recent years as "master" post-transcriptional regulators, we discuss the possibility that predominantly neuronal ELAVs (nELAVs) play multiple roles in regulating splicing, stability, localization, and translation of AChE mRNA.
 
Recent studies show a key role of brain inflammation in epilepsy. However, the mechanisms controlling brain immune response are only partly understood. In the periphery, acetylcholine (ACh) release by the vagus nerve restrains inflammation by inhibiting the activation of leukocytes. Recent reports suggested a similar anti-inflammatory effect for ACh in the brain. Since brain cholinergic dysfunctions are documented in epileptic animals, we explored changes in brain cholinergic gene expression and associated immune response during pilocarpine-induced epileptogenesis. Levels of acetylcholinesterase (AChE) and inflammatory markers were measured using real-time RT-PCR, in-situ hybridization and immunostaining in wild type (WT) and transgenic mice over-expressing the "synaptic" splice variant AChE-S (TgS). One month following pilocarpine, mice were video-monitored for spontaneous seizures. To test directly the effect of ACh on the brain's innate immune response, cytokines expression levels were measured in acute brain slices treated with cholinergic agents. We report a robust up-regulation of AChE as early as 48 h following pilocarpine-induced status epilepticus (SE). AChE was expressed in hippocampal neurons, microglia, and endothelial cells but rarely in astrocytes. TgS mice overexpressing AChE showed constitutive increased microglial activation, elevated levels of pro-inflammatory cytokines 48 h after SE and accelerated epileptogenesis compared to their WT counterparts. Finally we show a direct, muscarine-receptor dependant, nicotine-receptor independent anti-inflammatory effect of ACh in brain slices maintained ex vivo. Our work demonstrates for the first time, that ACh directly suppresses brain innate immune response and that AChE up-regulation after SE is associated with enhanced immune response, facilitating the epileptogenic process. Our results highlight the cholinergic system as a potential new target for the prevention of seizures and epilepsy.
 
Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway.
 
Schematic representation of the posttranslational processing of ghrelin. A signal peptide peptidase cleaves the signal sequence. Acylation of pro-ghrelin occurs by means of ghrelin O-acyl transferase (GOAT), which is located in the ER compartment and mediates the translocation of octanoyl-CoA. Once the pro-ghrelin precursor reaches the trans-Golgi compartment, it is cleaved by PC1/3 prohormone convertase. Different forms of ghrelin are released to the circulation: acylated, unacylated, and other shorter forms.
Summary of intracellular mechanisms mediating the neuroprotective effects of ghrelin. GHS–R1a activation result in release of intracellular calcium and protein kinase C (PKC) activation that leads to the stimulation of mitogen-activated protein kinases (MAPKs) pathway. The generation of phosphatidylinositol phosphates PIP3 and PIP2 induces the protein inositol 3 kinase (PI3K)/Akt pathway. MAPK and Akt stimulate cell proliferation and inhibit apoptosis. Ghrelin also regulates hypothalamic AMP-activated protein kinase (AMPK), phosphorylating (pAMPK), and activating it, which in turn phosphorylates and inactivates acetyl-CoA carboxylase (ACC), decreasing the cytoplasmic pool of malonyl-CoA, which promotes the generation of reactive oxygen species (ROS), which are buffered by uncoupling protein 2 (UCP-2).
Diagram summarizing the GHRP-6 survival actions proposed against monosodium glutamate (MSG) excitotoxicity. MSG activates JNK or p38, caspases and stimulates the translocation of apoptosis inducing factor (AIF). Growth hormone-releasing peptide (GHRP)-6 prevents cell death by inducing Bcl-2 and nuclear factor-kappa B (NF-κB) that results in the blockage of AIF translocation and caspase and PARP activation. Insulin-like growth factor (IGF)-I prevents cell death by blocking caspase activation.
The brain incorporates and coordinates information based on the hormonal environment, receiving information from peripheral tissues through the circulation. Although it was initially thought that hormones only acted on the hypothalamus to perform endocrine functions, it is now known that they in fact exert diverse actions on many different brain regions including the hypothalamus. Ghrelin is a gastric hormone that stimulates growth hormone secretion and food intake to regulate energy homeostasis and body weight by binding to its receptor, growth hormone secretagogues-GH secretagogue-receptor, which is most highly expressed in the pituitary and hypothalamus. In addition, ghrelin has effects on learning and memory, reward and motivation, anxiety, and depression, and could be a potential therapeutic agent in neurodegenerative disorders where excitotoxic neuronal cell death and inflammatory processes are involved.
 
Location of expanded repeats in disease genes. SCA, spinocerebellar ataxia (multiple loci numbered); FXTAS, fragile X tremor ataxia syndrome; ALS, amyotrophic lateral sclerosis; FTLD, frontotemporal lobar dementia; SBMA, spinobulbar muscular atrophy; DRPLA, dentatorubral-pallidoluysian atrophy.
Competing hypotheses of expanded repeat disease pathogenic pathways involving RNA. (1) RNA sequestration – via alternative splicing (Mankodi et al., 2002; Ranum and Day, 2004) or Akt/GSK3β pathway (van Eyk et al., 2011; Jones et al., 2012; Lawlor et al., 2012). (2) RAN (repeat associated non-AUG) Translation (Zu et al., 2011; Ash et al., 2013; Mori et al., 2013; Todd et al., 2013). (3) Toll “self” RNA recognition (Lawlor et al., 2011; Yu et al., 2011; Samaraweera et al., 2013).
Pathogenic mutations in Aicardi–Goutières syndrome. Mutations in genes in at least six distinct loci are able give rise to the constellation of symptoms that defines Aicardi–Goutières syndrome. Four of these (AGS2, AGS3, AGS4, and AGS6) are in genes that encode RNA-metabolizing proteins. The remaining two that have been identified (AGS1 and AGS5) are also in enzymes that have roles in nucleic acid metabolism. Deficiencies in any one of these enzymes are thought to result in the accumulation of endogenous nucleic acids that are sensed as “non-self” by Toll-like receptors, that in turn activate innate inflammatory pathways (Crow and Rehwinkel, 2009).
Hypothesis: expanded repeat neurodegenerative diseases are caused by the TLR recognition (and resultant innate inflammatory response) of repeat RNA as “non-self” due to their paucity of “self” modification that is exposed upon Dicer processing of double-strand RNA. Open circles represent sequence motifs for RNA modifying proteins; filled circles represent the modification of RNA at these specific sequence motifs (e.g., by methylation or A > I editing). Dicer is required for pathology in the Drosophila model and cleaves long high copy number repeat RNA down to 21mers [mainly r(CAG)7 mers; Lawlor et al., 2011]. These r(CAG)7 mers are, therefore, unmodified and recognized by TLRs as “non-self.” Toll-like receptor pathways (most probably the endosomal TLR3 receptor) are required for pathology (Samaraweera et al., 2013), through activation of the innate inflammatory pathway. Autophagy reduces pathology, possibly by metabolizing r(CAG)7 mers.
Repeat sequences that are expanded in copy number are the basis for ~20 dominantly inherited neurodegenerative diseases, including Huntington’s Disease. Despite some of the responsible genes being identified as long as 20 years ago, the identity and nature of the disease-causing pathogenic pathway remains a gap in knowledge for these diseases. This understanding is essential for rational approaches to delay onset, slow progression or ultimately effect cure. We have previously hypothesized that an RNA-based pathogenic pathway has a causal role in the dominantly inherited unstable expanded repeat neurodegenerative diseases. In support of this hypothesis we, and others, have characterized rCAG.rCUG100 repeat double-strand RNA (dsRNA) as a previously unidentified agent capable of causing pathogenesis in a Drosophila model of neurodegenerative disease. Dicer, Toll and autophagy pathways have distinct roles in this Drosophila dsRNA pathology. Dicer-dependence is accompanied by cleavage of rCAG.rCUG100 repeat double-strand RNA down to r(CAG)7 21-mers. Among the ‘molecular hallmarks’ of this pathway that have been identified in Drosophila, some [i.e. r(CAG)7 and elevated TNF] correlate with observations in affected people (e.g. HD, ALS) or in related animal models [i.e. autophagy]. The Toll pathway is activated in the presence of repeat-containing double-stranded RNA and toxicity is also dependent on this pathway. How might the endogenously expressed dsRNA mediate Toll-dependent toxicity in neuronal cells? Endogenous RNAs are normally shielded from Toll pathway activation as part of the mechanism to distinguish ‘self’ from ‘non-self’ RNAs. This typically involves post-transcriptional modification of the RNA. Therefore, it is likely that rCAG.rCUG100 repeat double-strand RNA has a characteristic property that interferes with or evades this normal mechanism of shielding. We predict that repeat expansion leads to an alteration in RNA structure and/or form that perturbs RNA mod
 
Calcium-activated chloride currents (CaCCs) are activated by an increase in intracellular calcium concentration. Peripheral nerve injury induces the expression of CaCCs in a subset of adult sensory neurons in primary culture including mechano- and proprioceptors, though not nociceptors. Functional screenings of potential candidate genes established that Best1 is a molecular determinant for CaCC expression among axotomized sensory neurons, while Tmem16a is acutely activated by inflammatory mediators in nociceptors. In nociceptors, such CaCCs are preferentially activated under receptor-induced calcium mobilization contributing to cell excitability and pain. In axotomized mechano- and proprioceptors, CaCC activation does not promote electrical activity and prevents firing, a finding consistent with electrical silencing for growth competence of adult sensory neurons. In favor of a role in the process of neurite growth, CaCC expression is temporally correlated to neurons displaying a regenerative mode of growth. This perspective focuses on the molecular identity and role of CaCC in axotomized sensory neurons and the future directions to decipher the cellular mechanisms regulating CaCC during neurite (re)growth.
 
Activating transcription factor 3 (ATF3) belongs to the ATF/cyclic AMP responsive element binding family of transcription factors and is often described as an adaptive response gene whose activity is usually regulated by stressful stimuli. Although expressed in a number of splice variants and generally recognized as a transcriptional repressor, ATF3 has the ability to interact with a number of other transcription factors including c-Jun to form complexes which not only repress, but can also activate various genes. ATF3 expression is modulated mainly at the transcriptional level and has markedly different effects in different types of cell. The levels of ATF3 mRNA and protein are normally very low in neurons and glia but their expression is rapidly upregulated in response to injury. ATF3 expression in neurons is closely linked to their survival and the regeneration of their axons following axotomy, and that in peripheral nerves correlates with the generation of a Schwann cell phenotype that is conducive to axonal regeneration. ATF3 is also induced by Toll-like receptor (TLR) ligands but acts as a negative regulator of TLR signaling, suppressing the innate immune response which is involved in immuno-surveillance and can enhance or reduce the survival of injured neurons and promote the regeneration of their axons.
 
Amino acid sequence alignment of selected NCS proteins (sequence numbering is for S.  pombe NCS-1). Secondary structure elements (helices and strands), EF-hand motifs (EF1 green, EF2 red, EF3 cyan, and EF4 yellow), and residues that interact with the myristoyl group (highlighted magenta) are indicated. Swiss Protein Database accession numbers are Q09711 (S. pombe NCS-1), Q06389 (S. cerevisiae Frq1), P21457 (bovine recoverin), and P43080 (human GCAP1).
Main chain structures of Ca2+-free myrisoylated NCS-1 (PDB ID: 212e) (A), recoverin (PDB ID: 1iku) (C), and GCAP1 (PDB ID: 2r2i) (E). Close-up views of the myristate binding pocket in NCS-1 (B), recoverin (D) and GCAP1 (F). EF-hands and myristoyl group (magenta) are colored as defined in Figure 1. Adapted from and originally published by Lim et al. (2011).
Structures of activator (A) vs. inhibitor forms (B) of GCAP1 adapted from and originally published by Lim et al. (2013).
Schematic diagram of retinal guanylyl cyclase activation by GCAP1. RetGC is shown in gray. The four EF-hands in GCAP1 are colored coded as defined in Figure 1, bound Mg2+ are depicted by blue circles, bound Ca2+ are orange circles, the C-terminal helix is white, and N-terminal myristoyl group is magenta. The labeled GCAP1 residues (K23, M26, G32, F73, V77, A78, and W94) have been implicated in the RetGC binding site (Lim et al., 2013; Peshenko et al., 2014). The binding of Ca2+ at EF4 causes conformational changes that are transmitted to the RetGC binding site by a Ca2+-myristoyl tug mechanism (see arrows) modified from Peshenko et al. (2012).
Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca(2) (+)-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca(2) (+)-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca(2) (+)-binding to both recoverin and NCS-1 cause large protein conformational changes that ejects the covalently attached myristoyl group into the solvent exterior and promotes membrane targeting (Ca(2) (+)-myristoyl switch). The GCAP proteins undergo much smaller Ca(2) (+)-induced conformational changes and do not possess a Ca(2) (+)-myristoyl switch. Recent structures of GCAP1 in both its activator and Ca(2) (+)-bound inhibitory states will be discussed to understand structural determinants that control their Ca(2) (+)-dependent activation of retinal guanylyl cyclases.
 
PI3K activation promotes the formation of synaptic contacts and dendritic spines, morphological features of glutamatergic synapses that are commonly known to be related to learning processes. In this report, we show that in vivo administration of a peptide that activates the PI3K signaling pathway increases spine density in the rat hippocampus and enhances the animals' cognitive abilities, while in vivo electrophysiological recordings show that PI3K activation results in synaptic enhancement of Schaffer and stratum lacunosum moleculare inputs. Morphological characterization of the spines reveals that subjecting the animals to contextual fear-conditioning training per se promotes the formation of large spines, while PI3K activation reverts this effect and favors a general change toward small head areas. Studies using hippocampal neuronal cultures show that the PI3K spinogenic process is NMDA-dependent and activity-independent. In culture, PI3K activation was followed by mRNA upregulation of glutamate receptor subunits and of the immediate-early gene Arc. Time-lapse studies confirmed the ability of PI3K to induce the formation of small spines. Finally, we demonstrate that the spinogenic effect of PI3K can be induced in the presence of neurodegeneration, such as in the Tg2576 Alzheimer's mouse model. These findings highlight that the PI3K pathway is an important regulator of neuronal connectivity and stress the relationship between spine size and learning processes.
 
Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). Our previous results showed that thymocytes isolated from stefin B-deficient mice are more sensitive to apoptosis induced by the protein kinase C (PKC) inhibitor staurosporin (STS) than the wild-type control cells. We have also shown that the increased expression of stefin B in the nucleus of T98G astrocytoma cells delayed cell cycle progression through the S phase. In the present study we examined if the nuclear or cytosolic functions of stefin B are responsible for the accelerated induction of apoptosis observed in the cells from stefin B-deficient mice. We have shown that the overexpression of stefin B in the nucleus, but not in the cytosol of astrocytoma T98G cells, delayed caspase-3 and -7 activation. Pretreatment of cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited caspase activation, while treatment with the inhibitor of calpains- and papain-like cathepsins (2S,3S)-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation. We concluded that the delay of caspase activation in T98G cells overexpressing stefin B in the nucleus is independent of cathepsin inhibition.
 
Schematic representation of the interrelated role of different early-life elements for the consequences of early-life adversity. Early-life adversities in the form of early-life stress, under/malnutrition and infection are known to modulate hippocampal development and altogether determine hippocampal structure and function in adulthood with adverse effects on learning and memory. During the early sensitive period of development the offspring is fully dependent on the mother. Maternal care encompasses several elements (sensory stimuli, transfer of nutrition, hormones, and antibodies). In fact it is mostly via disruption of maternal care (with exception of early-life infection which can directly act upon the offspring) that early adversities will elicit disruptions in hormonal, (neuro)inflammatory and nutritional profiles in the offspring. Because these elements affect one another, they will ultimately act synergistically to modulate hippocampal structure and function throughout life.
Early-life adversity increases the vulnerability to develop psychopathologies and cognitive decline later in life. This association is supported by clinical and preclinical studies. Remarkably, experiences of stress during this sensitive period, in the form of abuse or neglect but also early malnutrition or an early immune challenge elicit very similar long-term effects on brain structure and function. During early-life, both exogenous factors like nutrition and maternal care, as well as endogenous modulators, including stress hormones and mediator of immunological activity affect brain development. The interplay of these key elements and their underlying molecular mechanisms are not fully understood. We discuss here the hypothesis that exposure to early-life adversity (specifically stress, under/malnutrition and infection) leads to life-long alterations in hippocampal-related cognitive functions, at least partly via changes in hippocampal neurogenesis. We further discuss how these different key elements of the early-life environment interact and affect one another and suggest that it is a synergistic action of these elements that shapes cognition throughout life. Finally, we consider different intervention studies aiming to prevent these early-life adversity induced consequences. The emerging evidence for the intriguing interplay of stress, nutrition, and immune activity in the early-life programming calls for a more in depth understanding of the interaction of these elements and the underlying mechanisms. This knowledge will help to develop intervention strategies that will converge on a more complete set of changes induced by early-life adversity.
 
Models of p25 dysregulation in Alzheimer’s disease. (A) The first model implicating p25 in AD is shown. This model proposes that p25 expression is increased in AD. This increased expression is caused by amyloid oligomers that increase calcium signaling enhancing cleavage of p35 into p25. Increased p25 expression leads to an overactivation and mislocalization of Cdk5, which results in tau hyperphosphorylation, which is a prerequisite for neurofibrillary tangle formation and neurodegeneration. (B) Revised model of p25 dysregulation in AD. This model proposes that p25 expression is not increased but reduced in AD. This decreased expression is caused by amyloid oligomers that decrease NMDA receptor-dependent calcium signaling at the synapse (due to internalization and desensitization of NMDA receptors) impairing cleavage of p35 into p25. Decreased p25 expression reduces synthesis of particular synaptic proteins, which affects synaptogenesis, late LTP and memory formation.
About 15 years ago it was proposed that generation of the truncated protein p25 contributes to toxicity in Alzheimer's disease (AD). p25 is a calcium-dependent degradation product of p35, the principal activator of cyclin-dependent kinase 5 (Cdk5). The biochemical properties of p25 suggested that its generation would cause Cdk5 overactivation and tau hyperphosphorylation, a prerequisite for neurofibrillary tangle (NFT) formation. Whilst this model was appealing as it explained NFT formation, many laboratories could not confirm the finding of increased p25 generation in brain from AD patients. On the contrary, it emerged that p25 levels are reduced in AD. This reduction occurs primarily in the early stages of the disease. Further, p25 generation in the mouse hippocampus is associated with normal memory formation and p25 overexpression enhances synaptogenesis. Therefore, it transpires that p25 generation is a molecular memory mechanism that is impaired in early AD. I discuss the prospect that investigation of p25-regulated proteins will shed light into mechanisms underlying synaptic degeneration associated with memory decline in AD.
 
The biological function of the cholinesterase (ChE) enzymes has been studied since the beginning of the twentieth century. Acetylcholinesterase plays a key role in the modulation of neuromuscular impulse transmission in vertebrates, while in invertebrates pseudo cholinesterases are preeminently represented. During the last 40 years, awareness of the role of ChEs role in regulating non-neuromuscular cell-to-cell interactions has been increasing such as the ones occurring during gamete interaction and embryonic development. Moreover, ChE activities are responsible for other relevant biological events, including regulation of the balance between cell proliferation and cell death, as well as the modulation of cell adhesion and cell migration. Understanding the mechanisms of the regulation of these events can help us foresee the possible impact of neurotoxic substances on the environmental and human health.
 
All-trans retinoic acid (RA) plays important roles in brain development through regulating gene transcription. Recently, a novel post-developmental role of RA in mature brain was proposed. Specifically, RA rapidly enhanced excitatory synaptic transmission independent of transcriptional regulation. RA synthesis was induced when excitatory synaptic transmission was chronically blocked, and RA then activated dendritic protein synthesis and synaptic insertion of homomeric GluA1 AMPA receptors, thereby compensating for the loss of neuronal activity in a homeostatic fashion. This action of RA was suggested to be mediated by its canonical receptor RARα but no genetic evidence was available. Thus, we here tested the fundamental requirement of RARα in homeostatic plasticity using conditional RARα knockout (KO) mice, and additionally performed a structure-function analysis of RARα. We show that acutely deleting RARα in neurons eliminated RA's effect on excitatory synaptic transmission, and inhibited activity blockade-induced homeostatic synaptic plasticity. By expressing various RARα rescue constructs in RARα KO neurons, we found that the DNA-binding domain of RARα was dispensable for its role in regulating synaptic strength, further supporting the notion that RA and RARα act in a non-transcriptional manner in this context. By contrast, the ligand-binding domain (LBD) and the mRNA-binding domain (F-domain) are both necessary and sufficient for the function of RARα in homeostatic plasticity. Furthermore, we found that homeostatic regulation performed by the LBD/F-domains leads to insertion of calcium-permeable AMPA receptors. Our results confirm with unequivocal genetic approaches that RA and RARα perform essential non-transcriptional functions in regulating synaptic strength, and establish a functional link between the various domains of RARα and their involvement in regulating protein synthesis and excitatory synaptic transmission during homeostatic plasticity.
 
Juvenile mice do not respond behaviorally to acute ketamine treatment. (A) Four week old C57BL/6 male mice administered vehicle (saline) or ketamine (3 mg/kg) i.p. display similar latency to consume food in a novel environment. (B) Food consumption over a period of 5 min is indistinguishable between ketamine or vehicle-treated juveniles. (C) The same groups of mice tested 24 h after i.p. vehicle or ketamine treatment show comparable immobility in the forced swim test.
Ketamine application does not induce significant synaptic strength potentiation in juvenile animals. Field potentials (FPs) were recorded in control (n = 6) and ketamine-treated (20 μM) slices (n = 11), from 14-day-old rats. Initial FP slopes are plotted as a function of time (mean ± SEM). We did not observe significant changes of FP slopes compared to baseline after 90 min with or without application of ketamine (p = 0.097 and p = 0.24 respectively). Inset, Representative waveforms from control and ketamine-treated slices recorded at the times indicated by the numbers on the graph (1, 2).
Application of D-AP5 and ketamine at rest induces potentiation of AMPAR-mediated evoked neurotransmission. Field potentials (FPs) were recorded from ketamine and D-AP5 treated slices from adult animals. Initial FP slopes are plotted as a function of time (mean ± SEM).(A) Application of ketamine (20 μM) induces significant increase in FP slopes (n = 8, p < 0.05).(B) Application of D-AP5 (20 μM) induces significant increase in FP slopes (n = 9, p < 0.05). Insets (A,B), Representative waveforms from D-AP5 and ketamine-treated slices recorded at the times indicated by the numbers on the graph (1, 2).
Ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist that produces rapid antidepressant responses in individuals with major depressive disorder. The antidepressant action of ketamine has been linked to blocking NMDAR activation at rest, which inhibits eukaryotic elongation factor 2 kinase leading to desuppression of protein synthesis and synaptic potentiation in the CA1 region of the hippocampus. Here, we investigated ketamine mediated antidepressant response and the resulting synaptic potentiation in juvenile animals. We found that ketamine did not produce an antidepressant response in juvenile animals in the novelty suppressed feeding or the forced swim test. In addition ketamine application failed to trigger synaptic potentiation in hippocampal slices obtained from juvenile animals, unlike its action in slices from adult animals. The inability of ketamine to trigger an antidepressant response or subsequent synaptic plasticity processes suggests a developmental component to ketamine mediated antidepressant efficacy. We also show that the NMDAR antagonist AP5 triggers synaptic potentiation in mature hippocampus similar to the action of ketamine, demonstrating that global competitive blockade of NMDARs is sufficient to trigger this effect. These findings suggest that global blockade of NMDARs in developmentally mature hippocampal synapses are required for the antidepressant efficacy of ketamine.
 
A) Structure of myristoylated GCAP1 [Stephen et al. (2007)]. Myristoyl residue buried inside the EF-1/EF-2 pair of EF-hands is shown in red. (B) Dose-dependence of recombinant RetGC1 activation by myristoylated and G2A GCAP1 (mean ± SD). For details of the assay—see Materials and Methods.
The effect of myristoylation on co-localization of GCAP1 with RetGC in HEK293 cells. (A) GCAP1-GFP expressed in HEK293 cells without RetGC1 produces diffuse pattern spreading over the cytoplasm and the nucleus (Peshenko et al., 2008); a—fluorescence of GCAP1-GFP, b—same, but superimposed on DIC image of the cells, c—GCAP1 GFP fluorescence profile recorded across the cell along the black line in “b.” (B) GCAP1 GFP mutant, in which a conserved CysPro pair in EF-hand 1 loop required for interaction with RetGC is replaced by Gly (Hwang et al., 2004), was co-expressed with RetGC1 tagged at the N-terminus with mOrange variant of red fluorescent protein using protocol described in (Peshenko et al., 2008, 2011); notice that the diffuse pattern of GCAP1-GFP persists despite the presence of RetGC1. (C) Membrane localization of wild type GCAP1-GFP (green) co-expressed with RetGC1 (red); a—fluorescence of GCAP1-GFP, b—anti-RetGC1 immunofluorescence of AlexaFluor 568, c—GCAP1 GFP (green) and anti-RetGC1 (red) fluorescence profile recorded across the cell along the white line shown in “b”; the nuclei in “c” were counterstained with TO-PRO3 (pseudo-blue). (D) Same as C, but using G2A GCAP1-GFP mutant. (E) Distribution of GCAP1-GFP fluorescence between the membranes and the nucleus quantified as described in (Peshenko et al., 2008); each data point corresponds to an individual cell; ◦—GCAP1-GFP expressed alone, •—GCAP1-GFP co-expressed with RetGC1, •—G2A GCAP1-GFP co-expressed with RetGC1. (F) The GCAP1-GFP fluorescence distribution ratio (mean ± SEM) averaged from panel E demonstrates that the G2A GCAP1 mutant compartmentalizes with the RetGC1, although less efficiently than the wild type.
Ca2+ sensitivity of GCAP1 is affected by myristoylation.(A–D), Ca2+ binding isotherms obtained using fluorescent indicator dye BAPTA-2 titration protocol (Peshenko and Dizhoor, 2006). (A) Ca2+ binding by myristoylated D6S GCAP1 (◦), non-myristoylated WT GCAP1 (•), and non-myristoylated G2A GCAP1 (♦). (B) Comparison of the experimental data for Ca2+ binding by G2A GCAP1 (♦) with the theoretical curve for three-center binding model calculated using previously reported macroscopic association constants, 6.3 × 107, 5.0 × 106, and 2.0 × 103 M-1 (Dell'Orco et al., 2010) (- - -); the corresponding dissociation constants are shown next to each trace. (C,D) Change of the binding stoichiometry in non-myristoylated GCAP1 with one (D144N/D148G, C) or two (D100N/D102G/D144N/D148G, D) EF-hands inactivated. The data were fitted using two different models: panels (A,B)—by three-center binding model, Cabound/GCAP = (K1Caf + 2K1K2Ca2f + 3K1K2K3Ca3f)/(1 + K1Caf + K1K2Ca2f + K1K2K3Ca3f), where K1, K2, and K3 are macroscopic equilibrium constants; panels (C,D)—by simplified hyperbolic saturation function, (Cabound/GCAP) = Bmax × Cafree/(Cafree + Kd), where Cabound is the concentration of Ca2+ bound to GCAP1, calculated as Cabound = Catotal – Cafree, Bmax is mol of Ca2+ bound per mol of GCAP1 at saturation, Kd is the apparent dissociation constant. The data shown are representative from 3 to 5 independent experiments producing virtually identical results. (E) Normalized activity of the recombinant RetGC1 expressed in HEK293 cells reconstituted with 10 μM purified myristoylated GCAP1 (◦) or G2A GCAP1 (•) at different free Ca2+ concentrations and 1 mm free Mg2+. The activities in each series were normalized by the maximal activity in the corresponding series. The data were fitted by the equation, A = Amax+(Amax−Amin)/(1 + (Cafree/Ca1/2)n), where A is RetGC activity, Ca1/2 is the free Ca2+ concentration producing 50% effect and n is the Hill coefficient. For other conditions of the assay see Materials and Methods.
Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca(2+) concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca(2+) binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca(2+) binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca(2+) concentrations and so was Ca(2+) sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca(2+)-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.
 
Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. More than 90% of ALS cases are sporadic, and the majority of sporadic ALS patients do not carry mutations in genes causative of familial ALS; therefore, investigation specifically targeting sporadic ALS is needed to discover the pathogenesis. The motor neurons of sporadic ALS patients express unedited GluA2 mRNA at the Q/R site in a disease-specific and motor neuron-selective manner. GluA2 is a subunit of the AMPA receptor, and it has a regulatory role in the Ca(2+)-permeability of the AMPA receptor after the genomic Q codon is replaced with the R codon in mRNA by adenosine-inosine conversion, which is mediated by adenosine deaminase acting on RNA 2 (ADAR2). Therefore, ADAR2 activity may not be sufficient to edit all GluA2 mRNA expressed in the motor neurons of ALS patients. To investigate whether deficient ADAR2 activity plays pathogenic roles in sporadic ALS, we generated genetically modified mice (AR2) in which the ADAR2 gene was conditionally knocked out in the motor neurons. AR2 mice showed an ALS-like phenotype with the death of ADAR2-lacking motor neurons. Notably, the motor neurons deficient in ADAR2 survived when they expressed only edited GluA2 in AR2/GluR-B(R/R) (AR2res) mice, in which the endogenous GluA2 alleles were replaced by the GluR-B(R) allele that encoded edited GluA2. In heterozygous AR2 mice with only one ADAR2 allele, approximately 20% of the spinal motor neurons expressed unedited GluA2 and underwent degeneration, indicating that half-normal ADAR2 activity is not sufficient to edit all GluA2 expressed in motor neurons. It is likely therefore that the expression of unedited GluA2 causes the death of motor neurons in sporadic ALS. We hypothesize that a progressive downregulation of ADAR2 activity plays a critical role in the pathogenesis of sporadic ALS and that the pathological process commences when motor neurons express unedited GluA2.
 
Ten-4 expression in neurite outgrowth of Neuro-2a and PC12 cells. A) Morphology of Neuro-2a and PC12 cells during neurite outgrowth. Both Neuro-2a and PC12 cells formed and extended neurites 3 d after induction of neurite outgrowth in N2a medium and PC12 medium, respectively. Scale bar 20 m. B) Quantitative RT-PCR of Ten-4 in Neuro-2a and PC12 cells during neurite outgrowth in N2a medium and PC12 medium, respectively. Relative increase of Ten-4 mRNA expression in the Neuro-2a neurite outgrowth on d 3 was higher than that in PC12 cells. Ten-4 mRNA expression was normalized using GAPDH mRNA expression. Normalized Ten-4 expression in each cell on d 0 was set as 1.0. Error bars se. C) Semiquantitative RT-PCR of Ten-4 in Neuro-2a and PC12 cells during neurite outgrowth with both N2a and PC12 media. Neuro-2a expressed Ten-4 higher than PC12 on d 0 and 3 in both N2a and PC12 media. The induction of Ten-4 expression on d 3 was observed in either N2a or PC12 medium, while the expression level of the control-actin was not changed.
Expanded TNR induce altered mRNA splicing in TREDs.  Expanded CNG repeats sequester RBP in the nucleus that modulate exon usage during pre-mRNA maturation, including MBNL1, CELF1, p68/DDX5, hnRNP A2/B1, Pur a, Sam68. This results in altered alternative splicing in several genes, producing misslocalized or dysfunctional proteins that contribute to pathogenesis in TREDs.
Altered miRNA biogenesis in TREDs.  Expanded CNG repeats have affinity for key players of miRNA biogenesis such as DROSHA and DGCR8. Other proteins modulating the activity of DROSHA complex, such as MBNL1, p72 and p68, are also sequestered by expanded TNR. Functional depletion of these proteins leads to decreased production of mature miRNAs, which results in increased expression of miRNA targets.
Biogenesis and activity of sCNG in TREDs.  TNR expansions produce hairpin-like structures that are recognized and cleaved by Dicer to form small RNAs with repeated TNR (sCNG). Bidirectional transcription though the expanded TNR offers another source of sCNG, where perfectly complementary double stranded mRNAs with expanded TNR are cleaved by Dicer to form sCNG. sCNG are incorporated into the RISC complex and silence genes with partial or perfect complementarity.
Selective targeting of the expanded allele in TREDs.  shRNA, modified ASO or siRNA directed against the expanded allele results in degradation of mutant mRNA or blockage of the mutant protein synthesis, which impedes the detrimental activities of the expanded TNR in the mRNA and the mutant protein. With this strategy the normal allele is not targeted, allowing its function.
Trinucleotide-repeat expansion diseases (TREDs) are a group of inherited human genetic disorders normally involving late-onset neurological/neurodegenerative affectation. Trinucleotide-repeat expansions occur in coding and non-coding regions of unique genes that typically result in protein and RNA toxic gain of function, respectively. In polyglutamine (polyQ) disorders caused by an expanded CAG repeat in the coding region of specific genes, neuronal dysfunction has been traditionally linked to the long polyQ stretch. However, a number of evidences suggest a detrimental role of the expanded/mutant mRNA, which may contribute to cell function impairment. In this review we describe the mechanisms of RNA-induced toxicity in TREDs with special focus in small-non-coding RNA pathogenic mechanisms and we summarize and comment on translational approaches targeting the expanded trinucleotide-repeat for disease modifying therapies.
 
Drug addiction is a chronic, relapsing brain disorder which consists of compulsive patterns of drug-seeking and taking that occurs at the expense of other activities. The transition from casual to compulsive drug use and the enduring propensity to relapse is thought to be underpinned by long-lasting neuroadaptations in specific brain circuitry, analogous to those that underlie long-term memory formation. Research spanning the last two decades has made great progress in identifying cellular and molecular mechanisms that contribute to drug-induced changes in plasticity and behavior. Alterations in synaptic transmission within the mesocorticolimbic and corticostriatal pathways, and changes in the transcriptional potential of cells by epigenetic mechanisms are two important means by which drugs of abuse can induce lasting changes in behavior. In this review we provide a summary of more recent research that has furthered our understanding of drug-induced neuroplastic changes both at the level of the synapse, and on a transcriptional level, and how these changes may relate to the human disease of addiction.
 
Top-cited authors
Amit Maity
  • University of Pennsylvania
Jayashree Karar
  • Wistar Institute
Irina Vetter
  • The University of Queensland
Riccardo Brambilla
  • Cardiff University
Johan Tolö
  • University of Gothenburg