The β‐amyloid precursor protein (APP) has been extensively studied for its role as the precursor of the β‐amyloid protein (Aβ) of Alzheimer's disease. However, the normal function of APP remains largely unknown. This article reviews studies on the structure, expression and post‐translational processing of APP, as well as studies on the effects of APP in vitro and in vivo. We conclude that the published data provide strong evidence that APP has a trophic function. APP is likely to be involved in neural stem cell development, neuronal survival, neurite outgrowth and neurorepair. However, the mechanisms by which APP exerts its actions remain to be elucidated. The available evidence suggests that APP interacts both intracellularly and extracellularly to regulate various signal transduction mechanisms.
Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.
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.
Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.
Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.
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.
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.
Aggregate‐prone mutant proteins, such as α‐synuclein and huntingtin, play a prominent role in the pathogenesis of various neurodegenerative disorders; thus, it has been hypothesized that reducing the aggregate‐prone proteins may be a beneficial therapeutic strategy for these neurodegenerative disorders. Here, we identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL‐threo‐1‐Phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol and Genz‐123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT‐mammalian target of rapamycin (mTOR) signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on‐target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant α‐synuclein in neurons, it does so, according to our data, through autophagy‐independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease.
Intravenous immunoglobulin (IVIG) contains anti‐amyloid‐β antibodies as well as antibodies providing immunomodulatory effects that may modify chronic inflammation in Alzheimer's disease. Answers to important questions about IVIG transport into the central nervous system and assessments of any impact amyloid‐β has on this transport can be provided by in vitro models of the blood–brain barrier. In this study, amyloid‐β[1‐42] was pre‐aggregated into fibrillar or oligomeric structures, and various concentrations were incubated in the brain side of the blood–brain barrier model, followed by IVIG administration in the blood side at the therapeutically relevant concentrations of 5 and 20 mg/mL. IVIG accumulated in the brain side at physiologically relevant levels, with amyloid‐β pre‐incubation increasing IVIG accumulation. The increased transport effect was dependent on amyloid‐β structural form, amyloid‐β concentration, and IVIG dose. IVIG was found to decrease monocyte chemotactic protein‐1 levels 6.5–18% when low amyloid‐β levels were present and increase levels 4.2–23% when high amyloid‐β levels were present. Therefore, the presence, concentration, and structure of amyloid‐β plays an important role in the effect of IVIG therapy in the brain.
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.
An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.
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.
Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis induce misfolding and aggregation of the protein with the inherent propensity of mutant SOD1 to aggregate generally correlating, with a few exceptions, to the duration of illness in patients with the same mutation. One notable exception was the D101N variant, which has been described as wild‐type‐like. The D101N mutation is associated with rapidly progressing motor neuron degeneration but shows a low propensity to aggregate. By assaying the kinetics of aggregation in a well‐characterized cultured cell model, we show that the D101N mutant is slower to initiate aggregation than the D101G mutant. In this cell system of protein over‐expression, both mutants were equally less able to acquire Zn than WT SOD1. In addition, both of these mutants were equivalently less able to fold into the trypsin‐resistant conformation that characterizes WT SOD1. A second major difference between the two mutants was that the D101N variant more efficiently formed a normal intramolecular disulfide bond. Overall, our findings demonstrate that the D101N and D101G variants exhibit clearly distinctive features, including a different rate of aggregation, and yet both are associated with rapidly progressing disease.
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