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A Monoclonal Antibody to Amyloid Precursor Protein Induces Neuronal Apoptosis
Journal of Neurochemistry
Abstract
Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.
... Thus, Aβ is generated from APP by beta-and gamma-secretase and constitutes the core component of senile plaques. Robust evidence showed that sAPPα is a trophic factor having neurotrophic and neuroprotective activities [6][7][8]. A lack of sAPPα causes neuronal death [8][9][10] in cell culture and worsens human AD symptoms in clinical trials [11][12][13][14]. ...
... Robust evidence showed that sAPPα is a trophic factor having neurotrophic and neuroprotective activities [6][7][8]. A lack of sAPPα causes neuronal death [8][9][10] in cell culture and worsens human AD symptoms in clinical trials [11][12][13][14]. ...
... Aducanumab is a recombinant human IgG1 monoclonal antibody preferentially targeting the antigenic epitope of the amino acids 3-7 on the N-terminal region of Aβ peptide [15] which overlaps the epitope within the domain of sAP-Pα ( Figure 1). Laboratory studies showed that interfering with sAPPα expression by antisense oligonucleotides [9] or antibodies [8] a truncated [10] sAPPs in cell culture caused neuronal death. Additionally, infusing a vaccine or antibody against part or the whole length of Aβ involving an epitope within the sAPPα domain worsened symptoms in patients with AD [11][12][13][14]. ...
... To address this question we tested whether oligomerization of APP by 22C11 could recapitulate the neurotoxic effects of oligomeric Aβ. Previous reports have demonstrated that 22C11 is highly toxic to cultured cortical neurons (Rohn et al., 2000). Nevertheless, we had two objectives. ...
... The possibility remains however that these lower concentrations of 22C11 could be toxic after a longer exposure. As was previously reported (Rohn et al., 2000), we observed a significant amount of neuritic dystrophy in neurons exposed to toxic concentrations of 22C11 (Figure 4-5B), which was not apparent at the subapoptotic concentrations (data not shown). ...
... to dimerize APP. This technique had previously been utilized to study similar effects in cortical neurons (Mbebi et al., 2002;Rohn et al., 2000); however in our studies we focused primarily on hippocampal neurons, since the hippocampus is one of the region that is most affected in AD (reviewed by Burger, 2010). ...
... Lacking authentic ligands for APP, several groups subsequently showed that crosslinked antibodies against APP could induce Go activation, while hyperactivating this response in cultured neurons provoked calcium (Ca 2C ) overload, increased reactive oxygen species (ROS) levels, and induced apoptosis. [31][32][33] Moreover, mutant forms of APP that cause familial forms of AD were found to hyperactivate Gao and induce neurotoxic responses in cell culture, arguing that disease-associated isoforms of APP function as constitutively active Go-coupled receptors. 34 Notably, these neurotoxic effects were independent of Ab, 31 although subsequent work showed that Ab could also provoke APP-dependent cytotoxic responses that were blocked by pertussis toxin (PTX), a specific inhibitor of Gi/Go proteins. ...
... Initially, we treated neurons with crosslinked antibodies targeting the extracellular domain of APP (22C11) to mimic ligand activation (shown schematically in Fig. 1D), based on past reports that this method induced APP-Gao responses in transfected cells. 32,33,63 Although we consistently observed an increase in growth cone collapse/stall responses in these cultures, a caveat is that forced multimerization with crosslinked antibodies might induce pathological changes in APP-dependent responses, including caspase-associated regulation of secretase trafficking. 64 As an alternative, we used a new generation of anti-peptide polyclonal antibodies targeting different domains in APP (Aves Laboratories), which recognize both membrane-associated and intracellular pools of APP in mammalian neurons (unpublished observations). ...
Cleavage of the Amyloid Precursor Protein (APP) generates amyloid peptides that accumulate in Alzheimer Disease (AD), but APP is also upregulated by developing and injured neurons, suggesting that it regulates neuronal motility. APP can also function as a G protein-coupled receptor that signals via the heterotrimeric G protein Gαo, but evidence for APP-Gαo signaling in vivo has been lacking. Using Manduca as a model system, we showed that insect APP (APPL) regulates neuronal migration in a Gαo-dependent manner. Recently, we also demonstrated that Manduca Contactin (expressed by glial cells) induces APPL-Gαo retraction responses in migratory neurons, consistent with evidence that mammalian Contactins also interact with APP family members. Preliminary studies using cultured hippocampal neurons suggest that APP-Gαo signaling can similarly regulate growth cone motility. Whether Contactins (or other APP ligands) induce this response within the developing nervous system, and how this pathway is disrupted in AD, remains to be explored.
... The quantity of cell surface APP is tightly regulated by its very rapid turnover (57) and varies considerably between different cell types and in different activation states, independently of the total quantity of cellular APP (58,59). The reason for this tight regulation might be that the activation of cell surface APP is toxic under certain circumstances (19,20,(22)(23)(24). It is, therefore, essential to address the aggregation state of cell surface APP separate from total cellular APP. ...
... Although a large proportion of APP is cleaved in various cell compartments (55) and the fragments are secreted or degraded, a small proportion of transmembrane APP remains stably localized to the plasma membrane (70). Different lines of evidence have suggested both growth-promoting and toxic roles for transmembrane APP (19,20,(22)(23)(24)71), whereas there is unequivocal evidence for a neuroprotective role of sAPP␣ (14 -18). Both inappropriate signaling by transmembrane APP and a lowered sAPP␣ concentration may, therefore, contribute to neurodegeneration. ...
The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPalpha and the neurotoxic beta-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPalpha, as sAPPalpha binding disrupts APP dimers, and this disruption of APP dimers by sAPPalpha is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPalpha. These findings suggest a potentially beneficial effect of increasing sAPPalpha production or disrupting APP dimers for neuronal survival.
... In fact, it was found that crosslinking surface APP with monoclonal antibody 22C11, which mimics the interaction of Aβ to APP; enhances Aβ production by inactivating the adaptor protein Golgilocalized gamma ear-containing ARF-binding 3 (GGA3) preventing the sorting of BACE1 to lysosomes for degradation 56 . It is important to mention that, similar to toxic Aβ assemblies, multimerization of cell surface APP by incubation with antibody 22C11 also causes toxicity by activating Go protein signaling 57,58 . Altogether, these observations strengthen our observation and allow us to suggest that binding of Aβ assemblies to APP enhances its amyloidogenic (which was not certified by peer review) is the author/funder. ...
Alzheimers disease (AD) is characterized by a cognitive impairment associated to amyloid beta (Aβ) aggregation and deposition in the brain. Aβ is generated by sequential cleavage of the amyloid precursor protein (APP) by β–site APP cleaving enzyme 1 (BACE1) and γ–secretase complex. The mechanisms that underlie exacerbated production of Aβ, favoring its deposition in the brain, is largely unknown. In vitro studies have shown that Aβ aggregates trigger enhanced production of Aβ by a yet non described mechanism. Here, we show that in different cell types, including human neurons derived from induced pluripotent stem cells (iPSC), oligomers and fibrils of Aβ enhance the convergence and interaction of APP and BACE1 in endosomal compartments. We demonstrated a key role of Aβ–APP/Go/Gβγ signaling on the amyloidogenic processing of APP. We show that APP mutants with impaired capacity to bind Aβ or to activate Go protein, are unable to exacerbate APP and BACE1 colocalization in the presence of Aβ. Moreover, pharmacological inhibition of Gβγ subunits signaling with gallein, abrogate Aβ–dependent interaction of APP and BACE1 in endosomes preventing β–processing of APP. Collectively, these findings uncover a feed–forward mechanism of amyloidogenesis that might contribute to Aβ pathology in early stages of AD and suggest that gallein might have clinical relevance.
... To examine APP protein and its metabolites, we used first an antibody that recognizes the N terminus of the protein. 43 APP-FL produced a protein product consistent in size to that expected of APP ( Figure 1C). Expression of APPDex17 resulted in the production of a cellular protein that migrated faster than the APP-FL protein by gel electrophoresis, consistent with the deletion of the 49 amino acids encoded by exon 17 ( Figure 1C, left). ...
Alterations in amyloid beta precursor protein (APP) have been implicated in cognitive decline in Alzheimer’s disease (AD), which is accelerated in Trisomy 21/Down syndrome (DS), likely due to the extra copy of the APP gene, located on chromosome 21. Proteolytic cleavage of APP generates amyloid-β (Aβ) peptide, the primary component of senile plaques associated with AD. Reducing Aβ production is predicted to lower plaque burden and mitigate AD symptoms. Here, we designed a splice-switching antisense oligonucleotide (SSO) that causes skipping of the APP exon that encodes proteolytic cleavage sites required for Aβ peptide production. The SSO induced exon skipping in Down syndrome cell lines, resulting in a reduction of Aβ. Treatment of mice with the SSO resulted in widespread distribution in the brain accompanied by APP exon skipping and a reduction of Aβ. Overall, we show that an alternatively spliced isoform of APP encodes a cleavage-incompetent protein that does not produce Aβ peptide, and that promoting the production of this isoform with an SSO can reduce Aβ in vivo. These findings demonstrate the utility of using SSOs to induce a spliced isoform of APP to reduce Aβ as a potential approach for treating AD.
... Data are presented as the mean ± SEM of three independent experiments. ** p < 0.01 surface can transduce a cell signal through Go from extracellular ligands that trigger cell death [53,54]. s A P P α -m e d i a t e d r e s c u e f r o m t r o p h i c f a c t o r deprivation-induced cell death requires APP to transduce a G protein-mediated signal, leading to activation of the Akt survival pathway [55]. ...
Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble N-terminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPα-induced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.
... Coincidental with these observations, the overexpression of familial Alzheimer´s disease-forms of APP promotes constitutive Go activation leading to cellular toxicity (Niikura et al. 2004, Okamoto et al. 1995, Yamatsuji et al. 1996a, Yamatsuji et al. 1996b, Okamoto et al. 1996. Similarly, promoting cell-surface multimerization of APP with either, antibodies to APP-ectodomain or pathologic assemblies of Amyloid β also triggers Go activation and neuronal degeneration (Rohn et al. 2000, Sudo et al. 2000, Sola Vigo et al. 2009, Shaked et al. 2009, Xu et al. 2009). Although these observations provide compelling evidence that binding of pathogenic holo-APP ligands triggers Go activation and neurodegeneration in Alzheimer disease, the potential role of APP-Go signaling in the normal development and plasticity of the nervous system has received much less attention. ...
The amyloid precursor protein ( APP ) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell‐surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over‐expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. image
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... Subsequent investigations have generated an unexpectedly complicated (and often contradictory) view of how the APP- Go pathway might function in the diseased nervous system. Using a variety of transfected cell lines, Nishimoto et al. (1993) first confirmed that the induction of APP-Gαo signaling (by antibody crosslinking or induced dimerization) required transmembrane APP (Sudo et al., 2000; Hashimoto et al., 2003a), and that hyperactivation of this pathway could induce apoptotic responses in cultured mouse neurons (see also Rohn et al., 2000). Both groups described classic features of neuronal apoptosis in their assays, including neurite degeneration, nuclear condensation, internucleosomal DNA cleavage, and activation of pro-apoptotic caspases (including caspase 3, 7, and 9). ...
Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth via adenylyl cyclase/PKA-dependent pathways. These reports offer the intriguing perspective that G protein switching might modulate APP-dependent responses in a context-dependent manner. In this review, we provide an up-to-date perspective on the model that APP plays a variety of roles as an atypical G protein-coupled receptor in both the developing and adult nervous system, and we discuss the hypothesis that disruption of these normal functions might contribute to the progressive neuropathologies that typify AD.
... In this context, 22C11 could be considered to be an APP ligand mimetic of sorts. Indeed, sustained exposure of neurons to 22C11 causes neuronal apoptosis [20,38,39] and cell cycle entry [20] via a signal transduction pathway that is activated by C31 alone [20]. ...
... Die Initiation von Apoptose durch die Homodimiersierung von APP basiert u.a. auf Befunden von Rohn und Kollegen, die eine Aktivierung von APP mit nachfolgender Caspase-Aktivierung durch eine Präinkubation mit einem spezifischen APP-Antikörper (22C11) induzieren konnten(Rohn et al., 2000). Analog zur APP-Dimerisierung wird durch die Bindung des Antikörpers an den Oberflächenrezeptor APP die Auslösung einer Signalkaskade forciert, die in neuronalem Zelltod resultiert. ...
... Neuritic degeneration was observed followed by caspase dependent apoptosis when the monoclonal antibody binds to APP. This is suggestive of the involvement of APP in neuronal cell death in AD [75]. ...
Pharmacological studies reveal APP and Aβ have interactions with glutamate and calcium, cytokines, copper/zinc chelators, secretases and presenilins, nicotinic receptors, acetycholinesterase, neurotrophins, non-steroidal anti-inflame-matory drugs, monoclonal antibodies to Aβ, protease inhibitors, oestrogen, homocysteine, immediate early genes such as c-fos or c-jun and cholesterol. These functional and pharmacological observations highlight the need for greater understanding of APP and Aβ in brain function and have implications for clinical trials.
... APP at the cell surface is required for its cleavages. Beside its cleavages, APP can receive signals transduced through Go to induce neuronal migration and outgrowth, synapse differentiation at the neuromuscular junction in Drosophila or cell death [3][4][5][6][7][8][9][10]. The generation of APP cleavages following this signaling has not been reported. ...
Background
The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate.
Results
We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.
Conclusions
These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.
... Different lines of evidence suggest growth-promoting and neuroprotective roles for APP, but there are also reports linking APP to cell death. Exposure of rat cortical neurons to a monoclonal antibody directed against the extracellular domain of APP is shown to cause neurite degeneration followed by caspase dependent apoptosis (Rohn et al. 2000). The same year another group published similar results (Sudo et al. 2000). ...
... In neurons, APP and caspase-2 can regulate RhoA activation, and cells lacking either APP or caspase-2 are completely immune to Ab synaptotoxicity (Pozueta et al., 2013;Troy et al., 2000). In addition, dimerization of cellsurface APP by cross-linking cell-surface APP with a divalent antibody is sufficient to mimic the toxic effects of Ab in neurons (Lefort et al., 2012;Rohn et al., 2000). Interestingly, activation of RhoA in neuronal cells by lysophosphatidic acid (LPA) is also regulated by caspase-2 activity, suggesting that Ab and LPA share pathways (Pozueta et al., 2013). ...
Interference with microtubule stability by beta-amyloid peptide (Aβ) has been shown to disrupt dendritic function and axonal trafficking, both early events in Alzheimer's disease. However, it is unclear whether Aβ regulation of microtubule dynamics can occur independently of its action on tau. RhoA has been implicated in neurotoxicity by Aβ but the mechanism by which this activation generates cytoskeletal changes is also unclear. We found that oligomeric Aβ1-42 induced the formation of stable detyrosinated microtubules in NIH3T3 cells and this function resulted from the activation of a RhoA-dependent microtubule stabilization pathway regulated by integrin signaling and the formin mDia1. Induction of microtubule stability by Aβ was also initiated by APP-dimerization and required caspase activity, two previously characterized regulators of neurotoxicity downstream of Aβ. Finally, we found that this function was conserved in primary neurons and abolished by Rho inactivation, reinforcing a link between induction of stable detyrosinated microtubules and neuropathogenesis by Aβ. Our study reveals a novel activity of Aβ on the microtubule cytoskeleton that is independent of tau and associated with pathways linked to microtubule stabilization and Aβ-mediated neurotoxicity.
... That is, if AICD mediates a signaling event, its level should exhibit regulation. However, treating our stable APP-transfected CHO cells (7wB4) with agents known to regulate the ␣-secretase cleavage of the APP ectodomain and thus initiate RIP, e.g., phorbol esters (Buxbaum et al., 1990; Nitsch et al., 1992; Gabuzda et al., 1993; Hung et al., 1993) or crosslinking of the APP ectodomain with antibodies (Rohn et al., 2000; Sudo et al., 2000), did not noticeably alter the level of AICD in these non-neuronal cells (data not shown). We therefore asked whether a role for AICD-mediated signaling might be more readily detectable in neurons, because APP is highly expressed in neurons of organisms from Drosophila to humans (Coulson et al., 2000). ...
... For example, while Ab has been demonstrated to be highly toxic to cultured neurons 33 , APP itself appears to be necessary for this toxicity to occur 34 . The induction of APP signalling by its dimerization may in fact be sufficient to trigger neuronal death 35 . However, APP dimerization also leads to increased Ab production [36][37][38] and to the formation of an APP-derived cytosolic toxic fragment, C31 (refs 39-41). ...
Caspases have critical roles in Alzheimer's disease pathogenesis. Here we show that caspase-2 is required for the cognitive decline seen in human amyloid precursor protein transgenic mice (J20). The age-related changes in behaviour and dendritic spine density observed in these mice are absent when they lack caspase-2, in spite of similar levels of amyloid beta (Aβ) deposition and inflammation. A similar degree of protection is observed in cultured hippocampal neurons lacking caspase-2, which are immune to the synaptotoxic effects of Aβ. Our studies suggest that caspase-2 is a critical mediator in the activation of the RhoA/ROCK-II signalling pathway, leading to the collapse of dendritic spines. We propose that this is controlled by an inactive caspase-2/RhoA/ROCK-II complex localized in dendrites, which dissociates in the presence of Aβ, allowing for their activation and entry in the spine. These findings directly implicate caspase-2 as key driver of synaptic dysfunction in Alzheimer's disease and offer novel therapeutic targets.
... Together, these evidences suggest that APP-mediated alterations in Go signaling contribute to the toxic effects elicited by Aβ assemblies. Also, it was found that molecules that bind APP and promote its multimerization such as transforming growth factor β2 and APP-specific antibodies (i.e., 22C11) induce toxicity by a Go-dependent mechanism (Hashimoto et al., 2003(Hashimoto et al., , 2005Okamoto et al., 1995;Rohn et al., 2000;Sudo et al., 2000Sudo et al., , 2001. Additional evidence suggests that multimerization of holo-APP at the plasma membrane induces additional alterations in intracellular signaling. ...
A role of amyloid β (Aβ) peptide aggregation and deposition in Alzheimer's disease (AD) pathogenesis is widely accepted. Significantly, abnormalities induced by aggregated Aβ have been linked to synaptic and neuritic degeneration, consistent with the "dying-back" pattern of degeneration that characterizes neurons affected in AD. However, molecular mechanisms underlying the toxic effect of aggregated Aβ remain elusive. In the last 2 decades, a variety of aggregated Aβ species have been identified and their toxic properties demonstrated in diverse experimental systems. Concurrently, specific Aβ assemblies have been shown to interact and misregulate a growing number of molecular effectors with diverse physiological functions. Such pleiotropic effects of aggregated Aβ posit a mayor challenge for the identification of the most cardinal Aβ effectors relevant to AD pathology. In this review, we discuss recent experimental evidence implicating amyloid β precursor protein (APP) as a molecular target for toxic Aβ assemblies. Based on a significant body of pathologic observations and experimental evidence, we propose a novel pathologic feed-forward mechanism linking Aβ aggregation to abnormalities in APP processing and function, which in turn would trigger the progressive loss of neuronal connectivity observed early in AD.
... Patients who received AN-1792 have been reported to show elevated levels of antibodies against Aβ (Hock et al., 2002) and some immunized patients resulted in reduction of amyloid deposits without significant decrease in both NFT formation and neuronal cell death (Nicoll et al., 2003). As reported, there is a potential risk in the Aβ immunothrerapy that antibodies against APP induce neuronal cell death, which may lead to meningoencephalitis (Rohn et al., 2000; Sudo et al., 2000). Although it is reported that antibodies elevated in patients mainly recognize SPs, there is still a certain possibility that small fraction of elevated antibodies binds to APP and induces neuronal death. ...
... Prior studies using antibodies to cross-link APP have shown a significant amount of apoptotic death in cells exposed to high antibody concentrations (Rohn et al., 2000;Sudo et al., 2000). While neuronal death is invariably an important feature of AD, synaptic dysfunction is thought to be the earliest event in the progression of the disease (Delaère et al., 1989;Terry et al., 1991). ...
The accumulation of the β-amyloid peptide (Aβ) in Alzheimer's disease (AD) is thought to play a causative role in triggering synaptic dysfunction in neurons, leading to their eventual demise through apoptosis. Aβ is produced and secreted upon sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases. However, while Aβ levels have been shown to be increased in the brains of AD patients, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced, or whether the cleavage process changes over time. It has been proposed that Aβ can bind APP and promote amyloidogenic processing of APP, further enhancing Aβ production. Proof of this idea has remained elusive because a clear mechanism has not been identified, and the promiscuous nature of Aβ binding complicates the task of demonstrating the idea. To work around these problems, we used an antibody-mediated approach to bind and cross-link cell-surface APP in cultured rat primary hippocampal neurons. Here we show that cross-linking of APP is sufficient to raise the levels of Aβ in viable neurons with a concomitant increase in the levels of the β-secretase BACE1. This appears to occur as a result of a sorting defect that stems from the caspase-3-mediated inactivation of a key sorting adaptor protein, namely GGA3, which prevents the lysosomal degradation of BACE1. Together, our data suggest the occurrence of a positive pathogenic feedback loop involving Aβ and APP in affected neurons possibly allowing Aβ to spread to nearby healthy neurons.
... Recently, Rohn et al. (3) reported that treatment of primary cultured cortical neurons with anti-APP antibody causes cell death. We (4) independently found that anti-APP antibodies kill primary cultured cortical neurons as well as immortalized neuronal cells overexpressing APP 695 , but not parental cells. ...
Antibodies against APP, a precursor of Aβ deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Aβ mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Aβ40 and Aβ42 in the cultured media were undetectable by an assay capable of detecting 100 nM Aβ peptides. However, exogenously treated Aβ1-42 or Aβ1-43 required >3 μM to exert neurotoxicity, and 25 μM Aβ1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Aβ1-42, whereas serum attenuated toxicity by Aβ1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His657-Lys676, but not the Aβ1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Aβ.
... In a recent phase 2 trial of Bapineuzumab (a humanized anti-Ab monoclonal antibody) reversible vasogenic edema was detected via brain MRI in 10% of the treated patients (Salloway et al., 2009). Other concerns include autoimmune reactions elicited by anti-Ab antibodies binding to epitopes on CNS neurons (Rohn et al., 2000), and Fc-mediated microglia activation and phagocytosis of insoluble aggregates (Rogers and Shen, 2000). These adverse effects preclude the clinical use of anti-Ab immunotherapy. ...
Anti-beta-amyloid (Aβ) immunotherapy is effective in removing brain Aβ, but has shown to be associated with detrimental effects. We have demonstrated that Adeno-associated virus (AAV)-mediated delivery of an anti-Aβ single chain antibody (scFv) gene was effective in clearing brain Aβ without eliciting any inflammatory side effects in old APP(Swe)/PS1dE9 transgenic mice. In the present study, we tested the efficacy and safety of intramuscular delivery of the scFv gene in preventing brain Aβ deposition. The scFv gene was intramuscularly delivered to APP(Swe)/PS1dE9 transgenic mice at 3 months of age, prior to Aβ deposition in the brain. Six months later, we found that the transgenes were expressed in a stable form at the delivered sites, with a small amount of ectopic expression in the liver and olfactory bulb. Brain Aβ plaque formation, Aβ accumulation, AD-type pathologies and cognitive impairment were significantly attenuated in scFv-treated APP(Swe)/PS1dE9 transgenic mice relative to EGFP-treated mice. Intramuscular delivery of scFv gene was well tolerated by the animals, did not cause inflammation or microhemorrhage at the gene expression site and in the brain, and did not induce neutralizing antibodies in the animals. These findings suggest that peripheral application of scFv is effective and safe in preventing the development of Alzheimer's disease (AD), and would be a promising non-inflammatory immunological modality for prevention and treatment of AD.
... AβPP expression is also increased following brain injury and increased levels observed in apoptotic cells [58,113] . AβPP may also be involved in cell survival [97]. Although these facts may indicate the involvement of AβPP in neuroplasticity , the physiological functions of AβPP are not clear. ...
The use of stem cells for neuroreplacement therapy is no longer science fiction - it is science fact. We have succeeded in producing neural cells in the brain using both neural and mesenchymal stem cell transplantation and even systemic injection using a small molecular compound. We have seen the improvement of cognitive function in animal models following the application of these stem cell technologies. These results may promise a bright future for stem cell based neuroreplacement therapies for neurodegenerative diseases including Alzheimer's disease (AD). However, we have to consider the pathophysiological environments of individual diseases before clinical applications can be introduced. We must find the factors in the pathology that may affect stem cell biology and overcome the negative effects on neuroreplacement. Here, we discuss not only the potential for therapeutic applications of stem cell strategies in neuropathological conditions, but also how to overcome the adverse effects on the biology of stem cells due to the factors that are altered under AD pathology.
The book summarizes the role of multiple enzyme targets and strategies to design and develop novel drug candidates for Alzheimer's disease (AD). Insights from researchers across the globe from diverse fields are presented in a thematic volume.
The chapters highlight current information scientists have unraveled about the origin, pathogenesis and prevention of AD. The contributions consider both established and emerging drug targets viz. Tau proteins, TREM, and microglia. Topics covered in the book include multi-target anti-Alzheimer's agents, epigenetic modifications, and the role of specific proteins like TMP21 and Tau in AD. A section dedicated to pharmacological treatments discusses the significance of tubulin-modifying enzymes, memantine, and glutamate antagonists. Enzymatic targets for drug discovery are thoroughly examined, focusing on cholinesterase, secretases, and other enzymes. Additionally, the book explores innovative nano-carrier-based drug delivery methods, emphasizing the crucial role of nanotechnology in effective Alzheimer's treatment.
The book aims to inform students and researchers in the field of neuroscience, medicine and pharmacology about current research and biochemical nuances of AD pathogenesis and enzymatic drug targeting strategies.
There is growing research interest in the relationship between cancer and dementia. The necessity for innovative and efficient drug discovery methods is justified by the variety of the disease and the slow pace of therapeutic advancement in neurodegenerative disease. Drug repurposing is a revolutionary strategy that renews the traditional drug development process by demonstrating novel therapeutic applications for already approved medications. The idea of repurposing anticancer medications as innovative therapies has merit because of the shared molecular foundation and inverse tuning between cancer and neurodegeneration. There are numerous studies in the literature that highlight the success of anticancer medications as repurposed therapies. Among this the nuclear retinoid X receptor agonist bexarotene has coupled the restoration of cognitive ability (AD) by enhancing apoE expression in mice models of Alzheimer's disease. Recent investigations have demonstrated that treatment with bexarotene reduces the number of soluble forms of Aβ, particularly Aβ oligomers. Since then, bexarotene has been the subject of extensive discussion and numerous follow-up studies. Bexarotene is a special substance since the US-FDA has given it approval for use in other conditions, and there is evidence to support its mode of action in Alzheimer's disease. These results and bexarotene's emerging significance in the treatment of Alzheimer's dementia are discussed in this review. In order to create possible repurposed medications with no toxicity for neurodegenerative disorder, This review encourages more research to better understand the possible benefits of anticancer medications.
How dystrophic neurites form around amyloid plaques is a key aspect of understanding the early pathophysiology of Alzheimer's disease. At present, three hypotheses prevail: (1) dystrophies result from extracellular amyloid-beta (Aβ) toxicity; (2) dystrophies results from accumulation of Aβ into distal neurites; and (3) dystrophies represent blebbing of the somatic membrane of a neuron with high Aβ load. We utilized a unique feature of the common 5xFAD AD mouse model to test these hypotheses. Cortical layer 5 pyramidal neurons show intracellular APP and Aβ accumulation before amyloid plaque formation while dentate granule cells in these mice show no APP accumulation at any age. However, the dentate gyrus shows amyloid plaques by 3 months of age. By a careful confocal microscopic analysis we found no evidence of severe degeneration in amyloid laden layer 5 pyramidal neurons in contrast to hypothesis 3. Using injecting red fluorescent marker into lateral entorhinal projection neurons in 5xFAD mice with endogenous green fluorescent protein (GFP) in dentate granule cells we could demonstrate that all dystrophies is outer molecular layer originate from the axon terminal of entorhinal projection neurons. Immunostaining with vesicular glutamate transporter supported the axonal nature of the dystrophies in the acellular dentate molecular layer. We observed few small dystrophies in the GFP labeled granule cell dendrites. In general GFP labeled dendrites appear normal around the amyloid plaques. These findings favor hypothesis 2 as the most likely mechanism of dystrophic neurite formation.
The search for a clinically effective therapy for patients with Alzheimer's disease (AD) has been long and arduous. In some circles the recent US Food and Drug Administration (FDA) approval of the human monoclonal antibody, Aducanumab, was viewed as a welcome advance. However, the administrative decision, in the face of a negative review by the members of the FDA neurology advisory board raised many questions concerning its appropriateness. In response the FDA has modified the conditions under which the drug should be administered. Currently, the etiology of AD remains unknown. Thus, application of therapies based on the still controversial amyloid hypothesis deserves critical scrutiny. While successful animal studies based on the hypothesis have stimulated many clinical trials in humans, none of these have shown statistically clinical benefit, raising questions regarding the intrinsic validity of the hypothesis itself. However, each successive trial has benefited from the experiences of those which preceded it. Given these caveats, the relevance of an apparent beneficial response in a subset of Aducanumab treated study participants must be weighed carefully. There are competing hypotheses regarding the etiology and pathophysiology responsible for the development of AD, including tau protein aggregation, acetylcholine deficiency, neuroinflammation, among others, all of which remain controversial. Nonetheless, the newly approved agent, Aducanumab did show some subtle benefit in some mild AD patients. Understanding the current hypotheses and controversies may help better evaluate the limitations and challenges in anti-amyloid therapy and in exploration of more efficacious therapies in treating patients with AD in the future.
Amyloid β protein (Aβ) plays a critical role in pathogenesis of Alzheimer's disease (AD). Our previous studies indicated that the sequence 31-35 in Aβ molecule is an effective active center responsible for Aβ neurotoxicity in vivo and in vitro. In the present study, we prepared a novel antibody specifically targeting the sequence 31-35 of amyloid β protein, and investigated the neuroprotection of the anti-Aβ31-35 antibody against Aβ1-42 -induced impairments in neuronal viability, spatial memory, and hippocampal synaptic plasticity in rats. The results showed that the anti-Aβ31-35 antibody almost equally bound to both Aβ31-35 and Aβ1-42 , and pretreatment with the antibody dose-dependently prevented Aβ1-42 -induced cytotoxicity on cultured primary cortical neurons. In behavioral study, intracerebroventricular (i.c.v.) injection of anti-Aβ31-35 antibody efficiently attenuated Aβ1-42 -induced impairments in spatial learning and memory of rats. In vivo electrophysiological experiments further indicated that Aβ1-42 -induced suppression of hippocampal synaptic plasticity was effectively reversed by the antibody. These results demonstrated that the sequence 31-35 of Aβ may be a new therapeutic target, and the anti-Aβ31-35 antibody could be a novel immunotheraputic approach for the treatment of AD. This article is protected by copyright. All rights reserved.
Background: Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. Objective: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this aimed to express recombinant human single-chain variable antibody against amyloid β peptide. Design, time and setting: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006. Materials: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. Methods: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvA β construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPICgK-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvA 13 was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. Main outcome measures: Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA® sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. Results: Gene sequencing confirmed pPIC9K-scFvA β orientation. Recombinants were obtained by linearized pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. Conclusion: The expression vector pPIC9K-scFvA β was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.
In order to evaluate whether APP-Ab injection to hippocampus influence Morris water maze behavior and neuronal degeneration and to further investigate the potential mechanisms, rats were anaesthetized and fixed on a stereotaxic instrument and bilateral injection 1 μl of anti-APP antibody (10 g/L) was made using microsyringe. Meanwhile, NS or IgG-intrahippocampal-injected (1 μl; 10 g/L) rats served as vehicle controls. Antibodies were injected into the hippocampus (AP: -3.0; L and R: 2.0; V: 3.5 mm). The Morris water maze test was performed to test animals' learning and memory ability. After APP-Ab injection, cresyl violet and Fluoro-Jade B staining were used to investigate neuronal degeneration. Immunohistochemistry staining was used to detect MAP-2 and phosphorylated paxillin and tau distribution at hippocampus. APP-Ab injection to hippocampus could prolong the escape latency to find hidden platform and decreased the exploratory time and crossing numbers in the training quadrant. APP-Ab injection was also shown to cause neuronal cell death and degeneration by cresyl violet (CV) staining and Fluoro Jade-B (FJB) staining. Moreover, decreased MAP2 immunoreactivity, increased phosphorylated paxillin and phosphorylated tau immunostaining were observed in the pyramidal cells. It can be concluded that intrahippocampus injection of APP-Ab could induce cognitive deficits and neurodegenerative changes. APP-Ab injection also affected the distribution of MAP2, paxillin and tau protein.
Expression of a familial Alzheimer's disease ( AD )‐linked mutant of amyloid β precursor protein ( APP ) or the binding of transforming growth factor β2 to wild‐type (wt)‐ APP causes neuronal death by activating an intracellular death signal (a APP ‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro . These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD . A recent study has shown that the A673T mutation in the APP isoform APP 770 , corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP 695 (an AD ‐protective mutant of APP ), is linked to a reduction in the incidence rate of AD . Consistent with this, cells expressing the AD ‐protective mutant of APP produce less Aβ than cells expressing wt‐ APP . In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐ APP , but not in those expressing the AD ‐protective mutant of APP . This result suggests that the AD ‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP ‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP ‐mediated intracellular death signal. image
The A598T mutation of amyloid precursor protein APP is linked to a reduction in the incidence rate of Alzheimer's disease (AD). This study shows that TGFβ2 causes death in neuronal cells expressing wild‐type APP, but not in those expressing the AD‐protective mutant of APP, suggesting that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal.
IntroductionThe Beta Amyloid Precursor Protein (APP) and Beta Amyloid (Aβ) PeptideThe Presenilins (PSs)The Amyloid Cascade HypothesisAlternative HypothesesFuture Treatments for ADReferences
Alzheimer's DiseaseDementia with Lewy Bodies (DLB)Frontotemporal Lobar Degeneration (FTLD)Hippocampal SclerosisChronic Traumatic EncephalopathyVascular DementiaReferences
Beta-amyloid is a 40-42 amino acid peptide derived from the processing of the amyloid precursor protein. Beta-amyloid assumes many different conformations, which are capable of different biological activities. Yet, the in vivo role of Beta-amyloid in neuronal degeneration is still controversial, possibly because it can assume so many biological roles or personalities. In this chapter, we propose a multiple variable model for the role of Beta-amyloid and suggest that a major part of the controversy surrounding the role of Beta-amyloid in neurodegeneration and Alzheimer's disease is that it does not fit the single factor model that is so pervasive in current research.
Over the next 50 years the incidence of Alzheimer's disease (AD) in the United States is expected to triple from about 420,000 new cases in 1999 to 1.32 million per year. Assuming no successful intervention, the prevalence of AD could be expected to rise over the next 50 years by a factor of about 3.7–8.94 million, with the range of 4.55–15.81 million in the United States alone. With the exception of early-onset familial AD (FAD) the disease is age correlated and its incidence grows exponentially with age. In part, because of their increased longevity, women represent and will continue to represent the majority (68% in 1997) of the affected population. If one adds the year 2000 U.S. census figures to the population of the European Union (EU-15) and Japan, the prevalence of AD in “the major pharmaceutical markets” for that year could be estimated as close to 8.7 million. The as yet uncertain etiology of AD precluded the attempts to prevent its onset. Cholesterol, immune response, and oxidation damage appear to precede the deposition of amyloid β-peptides (Aβ) and formation of senile (neuritic) plaques and intracellular neurofibrillary tangles. This is why the epidemiology and in vitro studies fail to point to right therapies. The statins, antioxidants, anti-inflammatory agents, estrogens, or testosterone may belong to preventive measures but may fail as therapeutics. At present, no agent is available to arrest, delay, or reverse the progress of the disease.
Synapse and neuron losses are characteristic features ofAlzheimer’s disease (AD) and are believed to underlie the cognitive
impairments seen in this disorder. Amyloid β-protein (Aβ) is hypothesized to play a pivotal role in initiating AD pathogenesis,
but the precise mechanisms of Aβ-induced damage remain unclear. Caspases, a family of proteases best known for their role
in programmed cell death, may play a role in neurodegeneration. This review will focus on recent findings implicating a role
for caspase cleavage of the amyloid precursor protein (APP) in synaptic and neuronal injury in AD and how this pathway may
be initiated by the interaction of Aβ with APP.
The functioning and metabolic pathway of the amyloid β-precursor protein (APP) have not been fully elucidated. To fill this research gap, this study immunocytochemically investigated the intracellular localization of APP in the neuroblastoma cell line SK-N-SH and in normal primary cells. Using antibodies against the amino-terminal portion of the APP molecule, immunoreactivity was detected not only in the cytoplasm but also in the nucleus and nucleolus. Further analysis revealed the co-localization of amino acids 44-63 of the APP molecule with fibrillarin, a nucleolus marker. These findings indicate that a fraction of APP, including its amino-terminal portion, may be localized in the nucleus as well as in the nucleolus, suggesting an important role of APP in RNA metabolism and other intra-nucleolus functions.
Recent studies support the activation of apoptotic pathways in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so by either activation of a receptor-mediated pathway employing caspase-8 or through an alternative mitochondrial pathway involving oxidative stress. In the present study, the role of caspase-8 in the AD brain was examined by designing a caspase-cleavage site-directed antibody to one of the active fragments of caspase-8. In vitro analysis with this antibody, termed CASP-8p18, demonstrated that it recognized the active 18-kDa fragment of caspase-8 but not the precursor protein. In vivo immunohistochemical analysis using hippocampal tissue sections from AD or aged-matched control brains demonstrated CASP-8p18 immunolabeling of neurons in all AD cases, whereas little staining was observed in controls. These results were confirmed using a commercially available antibody that, like the CASP-8p18 antibody reacts only with the 18-kDa fragment of caspase-8 and not full-length caspase-8. As with CASP-8p18 antibody, the commercial antibody-labeled neurons in all AD cases, while showing a relative paucity of staining in representative control cases. Labeling of CASP-8p18 within tangle-bearing neurons was observed in double-labeling studies with AT8 or PHF-1, both markers for neurofibrillary tangles (NFTs). In addition, using a caspase-cleavage site-directed antibody that recognizes cleavage products of caspase-3 showed colocalization of this antibody with the CASP-8p18 antibody within NFTs. These results suggest a role for caspase-8 and the receptor-mediated apoptotic pathway as a mechanism leading to the activation of caspase-3 within neurons of the AD brain.
APP is a transmembrane precursor of β-amyloid, and its mutations cause early-onset familial Alzheimer's disease. We report a toxic function of normal wild-type APP (wtAPP). Treatment of neuronal F11 cells, immortalized embryonic day 13 neurons, overexpressing wtAPP with anti-APP antibodies caused death. Death was not induced by antibody in parental F11 cells. Death by antibody occurred through cell-surface APP, not through secreted APP, in a pertussis toxin-sensitive manner and was typical apoptosis, not observed in primary astrocytes or glioma cells overexpressing wtAPP, but observed in primary cortical neurons. Cell-surface APP thus performs a toxic function as an extracellularly controllable regulator of neuronal death. This study provides a novel insight into the normal and pathological functions of cell-surface wtAPP.
In this review, we argue that at least one insult that causes Alzheimer’s disease (AD) is disruption of the normal function of the amyloid precursor protein (APP). Familial Alzheimer’s disease (FAD) mutations in APP cause a disease phenotype that is identical (with the exception that they cause an earlier onset of the disease) to that of ‘sporadic’ AD. This suggests that there are molecular pathways common to FAD and sporadic AD. In addition, all individuals with Down syndrome, who carry an extra copy of chromosome 21 and overexpress APP several-fold in the brain, develop the pathology of AD if they live past the age of 40. These data support the primacy of APP in the disease. Although APP is the source of the β-amyloid (Aβ) that is deposited in amyloid plaques in AD brain, the primacy of APP in AD may not lie in the production of Aβ from this molecule. We suggest instead that APP normally functions in the brain as a cell surface signaling molecule, and that a disruption of this normal function of APP is at least one cause of the neurodegeneration and consequent dementia in AD. We hypothesize in addition that disruption of the normal signaling function of APP causes cell cycle abnormalities in the neuron, and that these abnormalities constitute one mechanism of neuronal death in AD. Data supporting these hypotheses have come from investigations of the molecular consequences of neuronal expression of FAD mutants of APP or overexpression of wild type APP, as well as from identification of binding proteins for the carboxyl-terminus (C-terminus) of APP.
Le vieillissement des populations est corrélé à l’augmentation des pathologies neurodégénératives liées à l’âge, plus particulièrement la maladie d’Alzheimer. La recherche de marqueurs précoces de la maladie ainsi que l’élaboration de nouvelles stratégies thérapeutiques constituent un enjeu de taille. Parmi les mécanismes moléculaires de la formation des plaques amyloïdes actuellement explorés, les formes oligomériques tronquées de peptide amyloïde (Aß), notamment le peptide Aß3(pE) 42 retrouvé à des stades précoces de la maladie, joueraient un rôle déterminant. Ces travaux de thèse ont permis de montrer, dans un premier temps, que l’injection intracérébrale de ce peptide chez la souris entraîne des altérations de la mémoire de travail et des capacités d’apprentissage, associées à une accumulation d’espèces réactives dérivées de l’oxygène dans des régions cérébrales spécifiques (hippocampe et bulbes olfactifs) de ces animaux. Des essais menés in vitro sur des cultures primaires de neurones de souris montrent leur implication dans les voies apoptotiques impliquant l’activation des caspases et la cascade métabolique de l’acide arachidonique. La seconde étape de ces travaux a constitué en l’étude des effets protecteurs d’un peptide antiapoptotique d’origine endogène, l’humanine (HN) et son variant S14G (HNG). In vitro, un effet protecteur de ces peptides a été mesuré après traitement de neurones en culture par le peptide Aß3(pE) 42. Les résultats les plus marquants résident dans les observations faites in vivo : en effet, ces peptides inhibent l’effet délétère de l’injection intracérébroventriculaire du peptide Aß3(pE) 42, en restaurant les performances mnésiques des animaux dans les tests comportementaux. A la lumière de ces résultats, les peptides HN pourraient constituer de nouveaux outils thérapeutiques dans le traitement ou la prévention des dommages cellulaires précoces liés à la présence des oligomères solubles du peptide Aß.
The biological function of full-length amyloid-beta protein precursor (APP), the precursor of Abeta, is not fully understood. Mounting studies reported that antibody binding to cell surface APP causes neuronal injury. However, the mechanism of cell surface APP mediating neuronal injury remains to be determined. Colocalization of APP with integrin on cell surface leads us to suppose that focal adhesion (FA) related mechanism is involved in surface APP-mediated neuronal injury. In the present study, results demonstrated that primary cultured neurons treated with antibody against APP-N-terminal not only caused neuronal injury and aberrant morphologic changes of neurite, but also induced reaction of FA proteins appearing an acute increase then decrease pattern. Moreover, the elevation of tyrosine phosphorylation of FA proteins including paxillin and focal adhesion kinase (FAK), and down-regulated expression of protein tyrosine phosphatase (PTP1B) induced by APP antibody were prevented by inhibitor of Src protein kinases 4-amino-5-(4-chlorophenyl)-7(t-butyl) pyrazol (3,4-D) pyramide (PP2) and G protein inhibitor pertussis toxin (PTX), implying that Src family kinase and G protein play roles in APP-induced FA signals. In addition, pretreatment with PTX and PP2 was able to suppress APP-antibody induced neuronal injury. Taken together, the results suggest a novel mechanism for APP mediating neuronal injury through deregulating FA signals.
Alzheimer's disease (AD) is characterized by Aβ overproduction and tau hyperphosphorylation. We report that an early, transient and site-specific AD-like tau hyperphosphorylation at Ser262 and Thr231 epitopes is temporally and causally related with an activation of the endogenous amyloidogenic pathway that we previously reported in hippocampal neurons undergoing cell death upon NGF withdrawal [Matrone, C., Ciotti, M.T., Mercanti, D., Marolda, R., Calissano, P., 2008b. NGF and BDNF signaling control amyloidogenic route and Ab production in hippocampal neurons. Proc. Natl. Acad. Sci. 105, 13138-13143]. Such tau hyperphosphorylation, as well as apoptotic death, is (i) blocked by 4G8 and 6E10 Aβ antibodies or by specific β and/or γ-secretases inhibitors; (ii) temporally precedes tau cleavage mediated by a delayed (6-12h after NGF withdrawal) activation of caspase-3 and calpain-I; (iii) under control of Akt-GSK3β-mediated signaling. Finally, we show that such site-specific tau hyperphosphorylation causes tau detachment from microtubules and an impairment of mitochondrial trafficking. These results depict, for the first time, a rapid interplay between endogenous Aβ and tau post-translational modifications which act co-ordinately to compromise neuronal functions in the same neuronal system, under physiological conditions as seen in AD brain.
The cleavage of amyloid precursor protein (APP) by caspases unmasks a domain extending from membrane to caspase cleavage site. This domain induces apoptosis in vitro and in vivo when overexpressed in neurons through the help of an internalization vector. In this model, we previously showed that SET rapidly binds to the internalized domain and is involved in downstream deleterious effects. Under these conditions SET mislocalizes from the nucleus to the cytoplasm, as in Alzheimer's disease (AD). In this report using the same model, we show that PAT1 attaches to the internalized domain earlier than SET and that this binding causes an increase in the levels of APP and APLP2 at the cell surface. Down regulation experiments of PAT1 and of APP and APLP2 show that the increase of the levels of APP and APLP2 at the cell surface triggers the cell death signal and SET mislocalization into the cytoplasm. In the context of AD these data suggest that mislocalization of SET into the cytoplasm may occur downstream of first cell death signal events involving PAT1 protein.
Altered glutathione metabolism in association with increased oxidative stress has been implicated in the pathogenesis of many diseases. However, whether strategies aimed at restoring glutathione concentration and homeostasis are effective in ameliorating or modifying the natural history of these states is unknown. In this review we discuss the pathogenic role for altered glutathione metabolism in such diseases as protein energy malnutrition, seizures, Alzheimer's disease, Parkinson's disease, sickle cell anaemia, chronic diseases associated with ageing and the infected state. In addition, we discuss the efficacy of glutathione precursors in restoring glutathione homeostasis both in vitro and in vivo.
Although evidence suggests that neurofibrillary tangles (NFTs) and neuronal cell loss are prominent features of Alzheimer's disease (AD), the relationship between the two remains unknown. In the present study, the relationship between the activation of apoptotic mechanisms and NFT formation in AD was investigated using a caspase-cleavage site-directed antibody to fodrin, an abundant neuronal cytoskeleton protein. This antibody recognized cleavage products of fodrin after digestion by caspase-3, but did not recognize full-length fodrin. In vitro analysis of this fodrin caspase-cleavage product (CCP) antibody demonstrates that it is a specific probe for the detection of apoptotic but not necrotic pathways in cultured neurons. To determine whether caspases cleave fodrin in vivo, tissue sections from controls and AD were immunostained for fodrin (CCPs). Although no staining was observed in control cases, labeling of neurons was observed in the hippocampus of all AD cases, which increased as a function of disease progression. To determine a possible relationship between caspase activation and NFT formation, double-labeling experiments with fodrin CCP and PHF-1 were performed. Co-localization of these markers was observed in many neurons, and quantitative analysis showed that as the extent of NFT formation increased, there was a significant corresponding increase in fodrin CCP immunolabeling (r = 0.84). Taken together, these results provide evidence for the activation of apoptotic mechanisms in neurons in the AD brain and suggest that there is an association between NFT formation and the activation of apoptotic pathways in AD.
Neurones in the brain produce beta-amyloid fragments from a larger precursor molecule termed the amyloid precursor protein (APP). When released from the cell, these protein fragments may accumulate in extracellular amyloid plaques and consequently hasten the onset and progression of Alzheimer's disease (AD). A beta fragments are generated through the action of specific proteases within the cell. Two of these enzymes, beta- and gamma-secretase, are particularly important in the formation of A beta as they cleave within the APP protein to give rise to the N-terminal and C-terminal ends of the A beta fragment, respectively. Consequently, many researchers are investigating therapeutic approaches that inhibit either beta- or gamma-secretase activity, with the ultimate goal of limiting A beta; production. An alternative AD therapeutic approach that is being investigated is to employ anti-A beta antibodies to dissolve plaques that have already formed. Both of these approaches focus on the possibility that accrual of A beta leads to neuronal degeneration and cognitive impairment characterised by AD and test the hypothesis that limiting A beta deposition in neuritic plaques may be an effective treatment for AD.
Amyloid beta precursor protein (APP) and prion protein (PrP) are cell membrane elements implicated in neurodegenerative diseases. Both proteins undergo endoproteolysis. Evidence is adduced from the literature hinting that the process in the two proteins could be related, their functions may overlap and their distributions coincide. It is proposed that PrP catalyses its own cleavage, the C-terminal fragment functions as an alpha secretase and the N-terminal segment chaperones the active site; the alpha secretase releases anticoagulant and neurotrophic ectodomains from APP. The proposals explain some features of spongiform encephalopathies.
In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of Go and G subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated Go or Gi induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495–689 of the -adrenergic receptor kinase-1, which blocks the specific functions of G. Co-transfection of G22 cDNAs, but not that of other Gxz (x = 1–3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G as a cell death mediator for the FAD-associated mutant of APP.
Membrane and cytoskeletal structures are known targets of oxidative injury. Brains from patients with Alzheimer's disease have cytoskeletal abnormalities and platelet and possible neuronal membrane lesions. The authors have recently demonstrated that superoxide anion is a powerful inducer of heat-shock protein synthesis, and have also shown that in response to oxidative stress or hyperthermia, intracellular levels of antioxidant enzymes increase to several folds. Whether the aforementioned mechanisms play a role in Alzheimer's disease has been suggested but is not totally established. While exploring this possibility, tissue sections from five brains with Alzheimer's disease and five neuropathologically normal age-matched controls were immunostained with polyclonal antibodies against superoxide dismutase (CuZn- and Mn- forms) and catalase. A standard avidin-biotin-peroxidase method was used for antigen detection. A subgroup of neurofibrillary tangles (15-25%) and senile plaques (50%) showed immunoreactivity for both enzymes with a staining pattern similar (but not identical) to that usually observed with antibodies against ubiquitin. Senile plaques displayed a granular pattern of immunostaining. Amyloid cores in mature classical plaques remained unstained. In addition, occasional elements with features consistent with reactive glial cells were strongly immunostained. Tangle-free neurons in both diseased and control brains showed weak to absent intracytoplasmic immunoreactivity. The immunoreactivity was totally abolished by preincubation of the primary antibodies with the corresponding purified antigens. These findings support the hypothesis that oxidative stress may be involved in the pathogenesis of Alzheimer's disease.
The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal- and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.
Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.
Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.
Alzheimer's disease is one of the prevalent forms of human dementia. Its pathology is distinguished by proteinaceous deposits ("amyloid") in the brain. They contain a peptide (beta A4) that is proteolytically derived from a larger transmembrane protein. To follow the different metabolic pathways of this Amyloid Precursor Protein (APP) may thus lead to the elucidation of the molecular basis of Alzheimer's disease. Specific antibodies are necessary tools for this task. Using synthetic peptides, we have characterized the epitope of the APP-specific monoclonal antibody 22C11; it is localized between residues 66 and 81 of APP. Some of the peptides flanking this site exhibited properties generally associated with amyloid, i.e. low solubility, filament formation, and birefringence after Congo Red staining. Exploiting differences in the peptides' aggregational properties, we present evidence that the two dyes Eosin and Direct Red 254, in conjunction with classical amyloid staining by Congo Red, can be used to characterize aggregating, amyloid-like peptides in vitro.
Amyloid β-protein (Aβ) is the major constituent of senile plaques and cerebrovascular amyloid deposits in Alzheimer's disease
and is proteolytically derived from its transmembrane parent protein the amyloid β-protein precursor (AβPP). Although the
physiological role(s) of secreted AβPPs are not fully understood, several potential functions have been described including
the regulation of hemostatic enzymes factors XIa and IXa and a role in cell adhesion. In the present study, we investigated
the proteolytic processing of AβPP by factor XIa (FXIa). Incubation of the human glioblastoma cell line U138 stably transfected
to overexpress the 695 isoform of AβPP with FXIa (2.5-5 nM) resulted in proteolytic cleavage of secreted AβPP. Higher concentrations
of FXIa (>25 nM) resulted in loss in cell adherence. Coincubation of FXIa with purified, recombinant Kunitz protease inhibitor
domain of AβPP blocked both the proteolytic processing of AβPP and the loss of cell adhesion. The RHDS cell adhesion site
of AβPP resides within residues 5-8 of the Aβ domain. Incubation of synthetic Aβ1-40 peptide with increasing concentrations of FXIa resulted in cleavage of Aβ between Arg5 and His6 within the cell adhesion domain of the peptide. FXIa-digested Aβ1-40 or AβPP695 lost their abilities to serve as cell adhesion substrates consistent with cleavage through this cell adhesion site. Together,
these results suggest a new potential biological function for FXIa in the modulation of cell adhesion. In addition, we have
shown that FXIa can proteolytically alter Aβ and therefore possibly modify its physiological and perhaps pathological properties.
Amyloid precursor protein (APP), a transmembrane precursor of beta-amyloid, possesses a function whereby it associates with G(o) through its cytoplasmic His657-Lys676. Here we demonstrate that APP has a receptor function. In phospholipid vesicles consisting of baculovirally made APP695 and brain trimeric G(o), 22C11, a monoclonal antibody against the extracellular domain of APP, increased GTP gamma S binding and the turnover number of GTPase of G(o) without affecting its intrinsic GTPase activity. This effect of 22C11 was specific among various antibodies and was observed neither in G(o) vesicles nor in APP695/Gi2 vesicles. In APP695/G(o) vesicles, synthetic APP66-81, the epitope of 22C11, competitively antagonized the action of 22C11. Monoclonal antibody against APP657-676, the G(o) binding domain of APP695, specifically blocked 22C11-dependent activation of G(o). Therefore, APP has a potential receptor function whereby it specifically activates G(o) in a ligand-dependent and ligand-specific manner.
beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.
Alzheimer's disease is characterized by the presence of senile plaques comprised primarily of deposits of the beta-amyloid protein (A beta) derived from larger amyloid precursor proteins (APP). We have identified a cDNA that encodes a 751-amino acid APP-like protein (designated APLP2) from the mouse that, with exception of the A beta region, is highly homologous to APP. In situ hybridization and quantitative polymerase chain reaction reveal that APLP2 and APP mRNA are expressed in similar, if not identical, neuronal populations and at similar levels. APLP2 appears to mature through the same unusual secretory/cleavage pathway as APP. Furthermore, widely utilized antibodies generated against non-overlapping epitopes of APP do not discriminate between APP and APLP2. Although APLP2 cannot give rise to A beta, its near identity to APP outside the A beta domain confounds the interpretation of previous immunocytochemical and biochemical characterizations of APP biosynthesis and metabolism.
The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.
The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.
Interleukin-1beta-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.
Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during ap optosis may have implications for the membrane blebbing seen during this process.
Loss of nerve cells is a hallmark of the pathology of Alzheimer's disease (AD), yet the patterns of cell death are unknown. By analyzing DNA fragmentation in situ we found evidence for cell death not only of nerve cells but also of oligodendrocytes and microglia in AD brains. In average, 30 times more brain cells showed DNA fragmentation in AD as compared to age-matched controls. Nuclear alterations suggestive of apoptosis were rare in degenerating cells. Even though the majority of degenerating cells were not located within amyloid deposits and did not contain neurofibrillary tangles, neurons situated within areas of amyloid deposits or affected by neurofibrillary degeneration revealed a higher risk of DNA fragmentation and death than cells not exposed to these AD changes.
: Recent data from several groups suggest that the primary mechanism of β-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert-butyl hydroperoxide) and β-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not β-amyloid peptides. Next, we examined whether β-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or β-amyloid. In a cell-free system, iron but not β-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by β-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to β-amyloid. The ability of β-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates β-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of β-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to β-amyloid that underlie its neurodegenerative actions.
Damage from free radicals has been demonstrated in susceptible neuronal populations in cases of Alzheimer disease. In this study, we investigated whether iron, a potent source of the highly reactive hydroxyl radical that is generated by the Fenton reaction with H2O2, might contribute to the source of radicals in Alzheimer disease. We found, using a modified histochemical technique that relies on the formation of mixed valence iron complexes, that redox-active iron is associated with the senile plaques and neurofibrillary tangles—the pathological hallmark lesions of this disease. This lesion-associated iron is able to participate in in situ oxidation and readily catalyzes an H2O2-dependent oxidation. Furthermore, removal of iron was completely effected using deferoxamine, after which iron could be rebound to the lesions. Characterization of the iron-binding site suggests that binding is dependent on available histidine residues and on protein conformation. Taken together, these findings indicate that iron accumulation could be an important contributor toward the oxidative damage of Alzheimer disease.
The amyloid beta protein (ABP) is a 40 to 42 amino acid peptide which accumulates in Alzheimer's disease plaques. It has been demonstrated that this peptide and a fragment derived from it are cytotoxic for cultured cortical nerve cells. It is shown here that ABP and an internal fragment encompassing residues 25 to 35 (beta 25-35) are cytotoxic to a clone of PC12 cells at concentrations above 1 x 10(-9)M and to several other cell lines at higher concentrations. Between 10(-9) and 10(-11) M beta 25-35 protects PC12 cells from glutamate toxicity. The antioxidant and free radical scavenger vitamin E inhibits ABP induced cell death. These results have implications regarding the prevention and treatment of Alzheimer's disease.
We present prospective clinical and neuropathologic details of a pedigree segregating familial Alzheimer's disease (FAD) associated with a mutation (G----A substitution) at nucleotide 2149 in exon 17 of the amyloid precursor protein (APP) gene. This mutation, which is predicted to cause the missense substitution of isoleucine for valine at codon 717 of APP, cosegregated perfectly with the FAD trait (lod score = 3.49 at theta = 0.00). The earliest clinical manifestations of the disease relate to deficits in memory function, cognitive processing speed, and attention to complex cognitive sets. These changes occurred in the absence of changes in nonmemory language and visuospatial functions. The neuropathologic features of FAD associated with the APP717 mutation in this family include severe neuronal loss, abundant neurofibrillary tangles, amyloid plaques, and amyloid angiopathy. These results provide independent confirmation that mutations in the APP gene are linked to the FAD trait in some families.
The beta A4 protein, the major component of the amyloid deposition characterizing Alzheimer's disease, derives from the amyloid protein precursor (APP), an integral membrane protein with soluble derivatives. The function of APP is unknown. Both soluble and membrane-associated human brain APP (10(-10) M) significantly increased (P less than 0.025) neurite length and branching in pheochromocytoma PC12 cells, but did not affect the number of neurites per cell. At higher concentrations, APP was cytotoxic, with a half-maximal concentration of 5 x 10(-9) M. Nerve growth factor (NGF) is known to affect APP expression in vivo and in vitro. Antibodies to APP specifically diminished the effects of NGF on neurite length and branching. Thus APP may act to mediate neurite outgrowth promotion by NGF.
The beta-amyloid precursor protein (APP) is a membrane-bound glycoprotein which has been proposed to play a role both as a growth factor and a mediator of cell adhesion. Using the Neuro-2A neuroblastoma cell line, we have investigated the capacity of APP to mediate neural cell adhesion. The cells express the protein at a high level, the immunohistochemical staining pattern at the level of the membrane having a punctate pattern. Fab' fragments of antibodies to the extracellular portion of the molecule were found to inhibit cell binding to a collagen substrate, but not to laminin, fibronectin, or poly-l-lysine. Fab' fragments of antibodies to the nerve cell adhesion molecule N-CAM also inhibited binding of Neuro-2A cells specifically to collagen. This inhibition of cell-surface binding was accompanied by a repression of neurite outgrowth in differentiating cells in the presence of antibodies. APP antibodies also inhibited neuron-neuron and neuron-glial binding, but not glial-glial cell adhesion. These data suggest that the APP, which is expressed primarily on differentiated neuronal cells, may play a role in the mediation of both cell-cell and cell-substrate adhesion.
Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.
An epitope-specific antibody directed against the first 16 amino acids of the beta amyloid protein (anti-BP16) immunoprecipitated the secreted form of the amyloid precursor protein (APP) from the conditioned medium of PC12 cells. This antibody caused neurite retraction in differentiated PC12 cells and inhibited cell-substratum adhesion in many neuronal and non-neuronal cell types. The inhibitory effect of anti-BP16 was abolished by preabsorption of the antibody with BP16 peptide. Antibodies directed against other domains of APP did not inhibit cell adhesion. The secreted form of APP may be important for cell adhesion in many different mammalian cell types.
beta-Amyloid peptide forms the senile plaques of Alzheimer's disease and has been previously demonstrated to have both trophic and toxic effects on neurons in vitro. We report here that synthetic beta-amyloid peptide shows both aggregation and neurotoxicity after a 2-4 day incubation period, but is neurite-promoting and not toxic in its initially solubilized state. SDS-PAGE characterization shows that newly solubilized beta-amyloid is predominantly monomeric whereas incubated peptide has several high molecular weight species.
To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.
Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.
The discovery of nonerythroid spectrin (Goodman et al., Proc. Natl. Acad. Sci. USA 78: 7570-7574, 1981) has generated interest equivalent to that occurring upon the identification of nonmuscle actin and myosin. Brain spectrin has become the best-studied member of the nonerythroid spectrin family of molecules. In this review, we discuss the structure and functional interactions of brain spectrin, as a prelude to attempting to resolve what are some of the more controversial questions in the field. We finish with a discussion of what may be the most profitable directions for future research.
In this brief review we discuss the structure, location, developmental expression and potential functions of the spectrin isoforms [spectrin(240/235) and spectrin (240/235E)] within mammalian brain. We also contrast the structure and location of mammalian and avian brain spectrin isoforms.
Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.
Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non-erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during apoptosis may have implications for the membrane blebbing seen during this process.
The use of cultured hippocampal slices for studies of calpain-mediated pathogenesis was investigated. Breakdown products (BDPs), which result from proteolysis of spectrin by calpain I, were assayed with BDP-specific antibodies developed against peptide sequences on either side of the calpain cleavage site. The antibodies recognized either amino- or carboxy-terminal BDPs (147-kD BDPN and 152-kD BDPC, respectively). Various pathogenic manipulations, including trimethyltin, certain snake venoms and agonists for excitatory amino acid receptors, were found to cause rapid and pronounced increases in the proteolytic fragments. These effects were selective, i.e., chemicals or toxins directed at nonglutamatergic neurons had little effect on BDP concentrations. Transient accumulations of spectrin fragments were obtained with brief applications of N-methyl-D-aspartate; longer infusions resulted in lasting increases. Results similar to these have been observed in vivo with ischemic episodes of varying duration. Agonists of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subclass of glutamate receptors produced significant increases in spectrin proteolysis; however, prolonged exposure of the slices to centrally active drugs that enhance the currents passed by the receptors did not. The sensitivity, selectivity and temporal properties of the proteolytic response support the idea that cultured slices can be used to analyze the events leading to and following from calpain activation in the adult brain.
Loss of nerve cells is a hallmark of the pathology of Alzheimer's disease (AD), yet the patterns of cell death are unknown. By analyzing DNA fragmentation in situ we found evidence for cell death not only of nerve cells but also of oligodendrocytes and microglia in AD brains. In average, 30 times more brain cells showed DNA fragmentation in AD as compared to age-matched controls. Nuclear alterations suggestive of apoptosis were rare in degenerating cells. Even though the majority of degenerating cells were not located within amyloid deposits and did not contain neurofibrillary tangles, neurons situated within areas of amyloid deposits or affected by neurofibrillary degeneration revealed a higher risk of DNA fragmentation and death than cells not exposed to these AD changes.
In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although
much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death
occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo
apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival
contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative
disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold
may have the potential to change the natural progression of some of these diseases.
The hypothesis that intense stimulation of NMDA receptors activates calpain was tested in long-term cultures of hippocampus. Slices prepared from 10-day-old rats were maintained for periods of up to 6 weeks and then assayed for a stable breakdown product that results from the proteolysis of spectrin by calpain. The breakdown product increased dramatically during the first 24 h after tissue preparation and then decreased to a low level that remained unchanged for weeks. NMDA caused a 2- to 3-fold increase in breakdown product that rose linearly with time (5-30 min) and was blocked by the receptor antagonist MK-801. The effect of NMDA was the same throughout the culture period and was dependent upon the concentration of extracellular calcium with no effect at 2 mM and maximal effect at 4 mM calcium. These results indicate that rapid activation of calpain occurs in undamaged hippocampal neurons following stimulation of NMDA receptors.
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When the mammalian proto-oncogene bcl-2 is overexpressed it can protect various types of cells both from normal and from experimentally induced apoptosis, but the molecular mechanisms involved are unknown. Although the Bcl-2 protein is membrane-associated, its subcellular location is controversial: two studies have suggested that it is mainly associated with the nuclear envelope and endoplasmic reticulum, whereas another study has suggested that it is mainly located in the inner mitochondrial membrane. The latter study has suggested that Bcl-2 might protect cells from apoptosis by altering mitochondrial function and that mitochondria may be involved in apoptosis. Here we report that human mutant cell lines that lack mitochondrial DNA (mtDNA), and therefore do not have a functional respiratory chain, can still be induced to die by apoptosis, and that they can be protected from apoptosis by the overexpression of bcl-2, suggesting that neither apoptosis nor the protective effect of bcl-2 depends on mitochondrial respiration. We also show that the Bcl-2 protein in overexpressing cells is associated with the nuclear envelope and endoplasmic reticulum, as well as with mitochondria.
The most characteristic change in progressive dementia of Alzheimer's type is a tissue deposit of amyloid beta/A4 protein, which is derived from its precursor protein APP (ref.2). Structural alterations of APP are implicated in the pathogenesis of Alzheimer's disease, but it is not known how they cause the disease. Although APP has a receptor-like architecture, is located on the neuronal surface, and has a conserved cytoplasmic domain, no receptor function has been demonstrated for APP. Here we report that APP forms a complex with G(o), a major GTP-binding protein in brain. The cytoplasmic APP sequence His 657-Lys 676 shows a specific G(o)-activating function and is necessary for complex formation. G(o) protein treated with GTP-gamma S lost the ability to associate with APP. This suggests that APP is a receptor coupled to G(o) and that abnormal APP-G(o) signalling is involved in the Alzheimer's disease process.
We immunocytochemically studied the expression of amyloid beta protein precursor (APP) in the brains of normal aged rats, and found APP accumulation in swollen neurites, most of which were axons. These swollen neurites appeared throughout the central nervous system of aged rats; most of them were negative for neurofilament, ubiquitin, and tau. Such widely distributed APP accumulation in swollen neurites may reflect impaired fast axonal transport due to aging. APP immunostaining may be a good method to detect widely distributed age-related changes.
APP is a transmembrane precursor of beta-amyloid. In dominantly inherited familial Alzheimer's disease (FAD), point mutations V6421, V642F and V642G have been discovered in APP695. Here we show that expression of these mutants (FAD-APPs) causes a clone of COS cells to undergo apoptosis associated with DNA fragmentation. Apoptosis by the three FAD-APPs was the highest among all possible V642 mutants; normal APP695 had no effect on apoptosis, suggesting that apoptosis by APP mutants in this system is phenotypically linked to the FAD trait. FAD-APP-induced apoptosis was sensitive to bcl-2 and most probably mediated by heteromeric G proteins. This study presents a model system allowing analysis of the mechanism for FAD-APP-induced cytotoxicity.
Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).
APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations V642I/F/G were discovered in APP695, which has been suggested by multiple studies to be a cell surface signaling receptor. We previously reported that normal APP695 encodes a potential GO-linked receptor with ligand-regulated function and that expression of the three FAD mutants (FAD-APPs), not normal APP, induces cellular outputs by GO-dependent mechanisms. This suggests that FAD-APPs are constitutively active GO-linked receptors. Here, we provide direct evidence for this notion. Reconstitution of either recombinant FAD-APP with GO vesicles induced activation of GO, which was inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD-APP. Consistent with the dominant inheritance of this type of FAD, this function was dominant over normal APP, because little activation was observed in APP695-GO vesicles. Experiments with antibody competition and sequence deletion indicated that His657-Lys676 of FAD-APP, which has been specified as the ligand-dependent GO-coupling domain of normal APP, was responsible for this constitutive activation, confirming that the three FAD-APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP to be a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors.
The loss of neurons by programmed cell death is a normal feature of the nervous system during development and has recently been implicated as a major mechanism of cell death in neurodegenerative diseases. In some cases, programmed cell death is induced by the activation of membrane receptors and is referred to as activation-induced programmed cell death. Activation-induced programmed cell death has been previously described in cells from the immune system, in which the activation of receptors by receptor clustering leads to programmed cell death. To determine whether activation-induced programmed cell death occurs in neurons, Concanavalin A was used to cross-link membrane receptors on cortical neurons. Concanavalin A-induced neuronal death was dose dependent and effective at concentrations previously shown to induce activation-induced programmed cell death in lymphocytes. Programmed cell death was attenuated when Concanavalin A-specific binding to neurons was blocked with methyl α-d-mannopyranoside. Succinyl Concanavalin A, which bound to Concanavalin A receptors but was ineffective at cross-linking them, did not induce programmed cell death. Concanavalin A-induced neuronal death exhibited many of the hallmarks associated with programmed cell death, such as membrane blebbing, nuclear condensation and margination, and internucleosomal DNA cleavage. In addition, neurons exposed to Concanavalin A displayed a rapid, robust, and persistent increase in the immediate early gene protein c-Jun. A similar increase in c-Jun precedes programmed cell death induced by β-amyloid in neurons, and under some conditions an increase in c-Jun has been shown to be required for programmed cell death to occur in neurons. Increased expression of c-jun and other immediate early genes has also been correlated with activation-induced programmed cell death in lymphocytes.
The total numbers of neurons with and without neurofibrillary changes in the hippocampal subdivisions were estimated in 16 subjects with Alzheimer disease (AD) and in 5 normal elderly controls. On the basis of clinical symptoms, AD patients were subdivided into relatively less (AD-1. Functional Assessment Staging [FAST] stages 7a to 7c) and more severely affected (AD-2, FAST stages 7e to 7f) patient groups. In the AD-1 group relative to controls, the total number of neurons was reduced only in CA1 and in the subiculum. In the AD-2 group, neuronal losses were found in all sectors of the cornu Ammonis and in the subiculum and ranged from 53% in CA3 to 86% in CA1. The dentate gyrus was the only hippocampal subdivision without significant neuronal loss. Within the combined AD patient groups, significant correlations were noted between both clinical stage and duration of AD and both the total number of neurons and the percentage of neurons with neurofibrillary changes in CA1, CA4, and the subiculum. Regression analyses predicted neuronal losses over the maximal observed duration of 22 years of 87% in CA1, 63% in CA4, and 77% in the subiculum. Our data suggest that over the course of AD, continuous neurofibrillary tangle formation and continuous neuronal loss occur in the hippocampal subdivisions. The rate of neuronal loss appears to be similar for CA1, CA4, and the subiculum.
The retinotectal projection in chick is a well studied model system for axon guidance. The 'stripe assay' provides a unique tool for investigating underlying molecules and mechanisms of axon guidance by non-diffusible substrate bound molecules in vitro. By combining this assay with a modified 'Campenot chamber', we have now investigated the role of several second messenger systems in this type of axon guidance by confronting growing axons with various drugs that are known to influence intracellular signaling. We have shown that extracellular, and most probably intracellular Ca++ is not required for this type of axon guidance, which also rules out the need for Ca++-dependent adhesion molecules like cadherins. While at least calmodulin and protein kinase C seem to be involved in axon elongation, inhibiting their function did not alter the growth cones' choice. Inhibition of other kinases, G-proteins and signaling components also failed to influence this guidance. These results may indicate that parallel signaling pathways take part in the molecular mechanism of this type of axon guidance.
Neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are a group of age-dependent,
progressive disorders that exhibit prominent neuronal death. Alzheimer’s disease and Parkinson’s disease are mainly sporadic,
whereas Huntington’s disease is entirely genetic. Studies on human postmortem brains highlighted the possible involvement
of apoptosis and autophagy in neuron death in the diseases. Studies using genetically engineered mouse models confirmed contributions
of key apoptosis genes in disease progression in these experimental systems. In addition, mouse models confirmed that neurotoxins
may accelerate and exacerbate disease progression. A better understanding of neuron death mechanisms in these diseases will
help design better treatment strategies.
The abnormal accumulation of β-amyloid (Aβ) in senile plaques appears to be a central pathological process in Alzheimer's disease. Aβ is formed by proteolysis of β-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and Aβ region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was ≃ 90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of Aβ is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.
Caspases (cysteinyl aspartate-specific proteinases) mediate highly specific proteolytic cleavage events in dying cells, which collectively manifest the apoptotic phenotype. The key and central role that these enzymes play in a biochemical cell-suicide pathway has been conserved throughout the evolution of multicellular eukaryotes.