A major hallmark feature of Alzheimer's disease is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ‐secretase complex and its activating protein (also known as γ‐secretase activating protein, or GSAP). Because GSAP interacts with the γ‐secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti‐Aβ therapy. GSAP derives from a C‐terminal fragment of a larger precursor protein of 98 kDa via a caspase 3‐mediated cleavage. However, the mechanism(s) involved in its degradation remain unknown. In this study, we show that GSAP has a short half‐life of approximately 5 h. Neuronal cells treated with proteasome inhibitors markedly prevented GSAP protein degradation, which was associated with a significant increment in Aβ levels and γ‐secretase cleavage products. In contrast, treatment with calpain blocker and lysosome inhibitors had no effect. In addition, we provide experimental evidence that GSAP is ubiquitinated. Taken together, our findings reveal that GSAP is degraded through the ubiquitin–proteasome system. Modulation of the GSAP degradation pathway may be implemented as a viable target for a safer anti‐Aβ therapeutic approach in Alzheimer's disease.
Vanishing white matter (VWM) is a recessive neurodegenerative disease caused by mutations in translation initiation factor eIF2B and leading to progressive brain myelin deterioration, secondary axonal damage, and death in early adolescence. Eif2b5R132H/R132H mice exhibit delayed developmental myelination, mild early neurodegeneration and a robust remyelination defect in response to cuprizone‐induced demyelination. In the current study we used Eif2b5R132H/R132H mice for mass‐spectrometry analyses, to follow the changes in brain protein abundance in normal‐ versus cuprizone‐diet fed mice during the remyelination recovery phase. Analysis of proteome profiles suggested that dysregulation of mitochondrial functions, altered proteasomal activity and impaired balance between protein synthesis and degradation play a role in VWM pathology. Consistent with these findings, we detected elevated levels of reactive oxygen species in mutant‐derived primary fibroblasts and reduced 20S proteasome activity in mutant brain homogenates. These observations highlight the importance of tight translational control to precise coordination of processes involved in myelin formation and regeneration and point at cellular functions that may contribute to VWM pathology.
Glutamate transport is a critical process in the brain that maintains low extracellular levels of glutamate to allow for efficient neurotransmission and prevent excitotoxicity. Loss of glutamate transport function is implicated in epilepsy, traumatic brain injury, and amyotrophic lateral sclerosis. It remains unclear whether or not glutamate transport can be modulated in these disease conditions to improve outcome. Here, we show that sirtuin (SIRT)4, a mitochondrial sirtuin, is up‐regulated in response to treatment with the potent excitotoxin kainic acid. Loss of SIRT4 leads to a more severe reaction to kainic acid and decreased glutamate transporter expression and function in the brain. Together, these results indicate a critical and novel stress response role for SIRT4 in promoting proper glutamate transport capacity and protecting against excitotoxicity.
Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre‐symptomatic (excitotoxin‐sensitive) transgenic mice expressing full‐length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex‐NMDAR) localization and signaling. Furthermore, Ex‐NMDAR stimulation facilitates excitotoxicity in wild‐type cortical neurons via calpain‐mediated cleavage of STriatal‐Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen‐activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain‐mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal‐regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6‐week‐old, excitotoxin‐sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA‐induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR‐post‐synaptic density protein‐95 (PSD‐95) binding but not by calpain inhibition. In 1‐year‐old excitotoxin‐resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR–PSD‐95 interactions contributes to elevated p38 signaling in early, excitotoxin‐sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin‐resistant stages.
Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca2+‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.
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.
Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.
Metabotropic glutamate receptor 5 (mGluR5) regulates excitatory post‐synaptic signaling in the central nervous system (CNS) and is implicated in various CNS disorders. Protein kinase A (PKA) signaling is known to play a critical role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia, and addiction. Dopamine signaling is known to modulate the properties of mGluR5 in a cAMP‐ and PKA‐dependent manner, suggesting that mGluR5 may be a direct target for PKA. Our study identifies mGluR5 at Ser870 as a direct substrate for PKA phosphorylation and demonstrates that this phosphorylation plays a critical role in the PKA‐mediated modulation of mGluR5 functions such as extracellular signal‐regulated kinase phosphorylation and intracellular Ca2+ oscillations. The identification of the molecular mechanism by which PKA signaling modulates mGluR5‐mediated cellular responses contributes to the understanding of the interaction between dopaminergic and glutamatergic neuronal signaling.
For over the last 50 years, the molecular mechanism of anti‐psychotic drugs' action has been far from clear. While risperidone is very often used in clinical practice, the most efficient known anti‐psychotic drug is clozapine (CLO). However, the biochemical background of CLO's action still remains elusive. In this study, we performed comparative proteomic analysis of rat cerebral cortex following chronic administration of these two drugs. We observed significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins caused by both antipsychotics. Among other proteins, alterations in collapsin response mediator proteins, CRMP2 and CRMP4, were the most spectacular consequences of treatment with both drugs. Moreover, risperidone increased the level of proteins involved in cell proliferation such as fatty acid‐binding protein‐7 and translin‐associated factor X. CLO significantly up‐regulated the expression of visinin‐like protein 1, neurocalcin δ and mitochondrial, stomatin‐like protein 2, the calcium‐binding proteins regulating calcium homeostasis, and the functioning of ion channels and receptors.
The parkin‐associated endothelial‐like receptor (PAELR, GPR37) is an orphan G protein‐coupled receptor that interacts with and is degraded by parkin‐mediated ubiquitination. Mutations in parkin are thought to result in PAELR accumulation and increase neuronal cell death in Parkinson's disease. In this study, we find that the protein interacting with C‐kinase (PICK1) interacts with PAELR. Specifically, the Postsynaptic density protein‐95/Discs large/ZO‐1 (PDZ) domain of PICK1 interacted with the last three residues of the c‐terminal (ct) located PDZ motif of PAELR. Pull‐down assays indicated that recombinant and native PICK1, obtained from heterologous cells and rat brain tissue, respectively, were retained by a glutathione S‐transferase fusion of ct‐PAELR. Furthermore, coimmunoprecipitation studies isolated a PAELR‐PICK1 complex from transiently transfected cells. PICK1 interacts with parkin and our data showed that PICK1 reduces PAELR expression levels in transiently transfected heterologous cells compared to a PICK1 mutant that does not interact with PAELR. Finally, PICK1 over‐expression in HEK293 cells reduced cell death induced by PAEALR over‐expression during rotenone treatment and these effects of PICK1 were attenuated during inhibition of the proteasome. These results suggest a role for PICK1 in preventing PAELR‐induced cell toxicity.
The role of physical exercise as a neuroprotective agent against ischemic injury has been extensively discussed. Nevertheless, the mechanisms underlying the effects of physical exercise on cerebral ischemia remain poorly understood. Here, we investigate the hypothesis that physical exercise increases ischemic tolerance by decreasing the induction of cellular apoptosis and glutamate release. Rats (n = 50) were submitted to a swimming exercise protocol for 8 weeks. Hippocampal slices were then submitted to oxygen and glucose deprivation. Cellular viability, pro‐apoptotic markers (Caspase 8, Caspase 9, Caspase 3, and apoptosis‐inducing factor), and glutamate release were analyzed. The percentage of cell death, the amount of glutamate release, and the expression of the apoptotic markers were all decreased in the exercise group when compared to the sedentary group after oxygen and glucose deprivation. Our results suggest that physical exercise protects hippocampal slices from the effects of oxygen and glucose deprivation, probably by a mechanism involving both the decrease of glutamatergic excitotoxicity and apoptosis induction.
Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal‐associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA‐transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.
Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.
Parkinson's disease (PD) is an age‐related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α‐synuclein‐containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN‐induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early‐onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy‐lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2‐D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6‐month‐old PINK1‐deficient mice and wild‐type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD.
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