Histone deacetylase mediates the decrease in drebrin cluster density induced by amyloid beta oligomers

Dendritic spine defects are found in a number of cognitive disorders, including Alzheimer’s disease (AD). Amyloid beta (Aβ) toxicity is mediated not only by the fibrillar form of the protein, but also by the soluble oligomers (Aβ-derived diffusible ligands, ADDLs). Drebrin is an actin-binding protein that is located at mature dendritic spines. Because drebrin expression is decreased in AD brains and in cultured neurons exposed to Aβ, it is thought that drebrin is closely associated with cognitive functions. Recent studies show that histone deacetylase (HDAC) activity is elevated in the AD mouse model, and that memory impairments in these animals can be ameliorated by HDAC inhibitors. In addition, spine loss and memory impairment in HDAC2 over-expressing mice are ameliorated by chronic HDAC inhibitor treatment. Therefore, we hypothesized that the regulation of histone acetylation/deacetylation is critical to synaptic functioning. In this study, we examined the relationship between HDAC activity and synaptic defects induced by ADDLs using an HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). We show that ADDLs reduce the cluster density of drebrin along dendrites without reducing drebrin expression. SAHA markedly increased the acetylation of histone proteins, and it simultaneously attenuated the ADDL-induced decrease in drebrin cluster density. In comparison, SAHA treatment did not affect the density of drebrin clusters or dendritic protrusions in control neurons. Therefore, SAHA likely inhibits ADDL-induced drebrin loss from dendritic spines by stabilizing drebrin in these structures, rather than by increasing drebrin clusters or dendritic protrusions. Taken together, our findings suggest that HDAC is involved in ADDL-induced synaptic defects, and that the regulation of histone acetylation plays an important role in modulating actin cytoskeletal dynamics in dendritic spines under cellular stress conditions, such as ADDL exposure.     

In vivo hippocampal microdialysis reveals impairment of NMDA receptor–cGMP signaling in APPSW and APPSW/PS1L166P Alzheimer’s transgenic mice

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer’s disease (AD) including memory deficit, presence of β-amyloid (Aβ) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-d-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Aβ oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15 months old APP_SW and 4 months old APP_SW/PS1_L166P Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Aβ oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5 ± 3.6 ng/g in 4 months old APP_SW mice to 60.8 ± 15.9 ng/g in 4 months old APP_SW/PS1_L166P mice. Four months old APP_SW mice had no Aβ amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aβ amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Aβ oligomers but predating onset of Aβ amyloidosis.     

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin.

The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.     

Abeta toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets

Over the past several years, experiments with synthetic amyloid-beta peptide (Abeta) and animal models have strongly suggested that pathogenesis of Alzheimer's disease (AD) involves soluble assemblies of Abeta peptides (Trends Neurosci. 24 (2001) 219). These soluble neurotoxins (known as ADDLs and protofibrils) seem likely to account for the imperfect correlation between insoluble fibrillar amyloid deposits and AD progression. Recent experiments have detected the presence of ADDLs in AD-afflicted brain tissue and in transgenic-mice models of AD. The presence of high affinity ADDL binding proteins in hippocampus and frontal cortex but not cerebellum parallels the regional specificity of AD pathology and suggests involvement of a toxin receptor-mediated mechanism. The properties of ADDLs and their presence in AD-afflicted brain are consistent with their putative role even in the earliest stages of AD, including forms of mild cognitive impairment.     

Subunit-specific NMDA receptor trafficking to synapses

To elucidate mechanisms controlling the number and subunit composition of synaptic NMDA-Rs in hippocampal slice neurons, the NR1, NR2A, and NR2B subunits were optically and electrophysiologically tagged. The NR2 subunit directs delivery of receptors to synapses with different rules controlling NR2A and NR2B. Synaptic incorporation of NR2B-containing receptors is not limited by synaptic transmission nor enhanced by increased subunit expression. NR2A-containing receptors whose expression normally increases with age replace synaptic NR2B-containing receptors. Replacement is enhanced by increased NR2A expression and requires synaptic activity. Surprisingly, spontaneously released transmitter acting on synaptic NMDA-Rs is sufficient for replacement and reduces NMDA-R responses. Thus, as with AMPA-Rs, synaptic trafficking of NMDA-Rs is tightly regulated and has subunit-specific rules with functionally important consequences.     

Inhibition of Calcineurin-mediated Endocytosis and ��-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Prevents Amyloid �� Oligomer-induced Synaptic Disruption

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid β (Aβ) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Aβ oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.     

Monoclonal antibodies that target pathological assemblies of Abeta

Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.     

Possible implications of insulin resistance and glucose metabolism in Alzheimer's disease pathogenesis

Type 2 diabetes mellitus (DM) appears to be a significant risk factor for Alzheimer disease (AD). Insulin and insulin-like growth factor-1 (IGF-1) also have intense effects in the central nervous system (CNS), regulating key processes such as neuronal survival and longevity, as well as learning and memory. Hyperglycaemia induces increased peripheral utilization of insulin, resulting in reduced insulin transport into the brain. Whereas the density of brain insulin receptor decreases during age, IGF-1 receptor increases, suggesting that specific insulin-mediated signals is involved in aging and possibly in cognitive decline. Molecular mechanisms that protect CNS neurons against β-amyloid-derived-diffusible ligands (ADDL), responsible for synaptic deterioration underlying AD memory failure, have been identified. The protection mechanism does not involve simple competition between ADDLs and insulin, but rather it is signalling dependent down-regulation of ADDL-binding sites. Defective insulin signalling make neurons energy deficient and vulnerable to oxidizing or other metabolic insults and impairs synaptic plasticity. In fact, destruction of mitochondria, by oxidation of a dynamic-like transporter protein, may cause synapse loss in AD. Moreover, interaction between Aβ and τ proteins could be cause of neuronal loss. Hyperinsulinaemia as well as complete lack of insulin result in increased τ phosphorylation, leading to an imbalance of insulin-regulated τ kinases and phosphatates. However, amyloid peptides accumulation is currently seen as a key step in the pathogenesis of AD. Inflammation interacts with processing and deposit of β-amyloid. Chronic hyperinsulinemia may exacerbate inflammatory responses and increase markers of oxidative stress. In addition, insulin appears to act as 'neuromodulator', influencing release and reuptake of neurotransmitters, and improving learning and memory. Thus, experimental and clinical evidence show that insulin action influences cerebral functions. In this paper, we reviewed several mechanisms by which insulin may affect pathophysiology in AD.     

NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII

Calcium entry through postsynaptic NMDA-Rs and subsequent activation of CaMKII trigger synaptic plasticity in many brain regions. Active CaMKII can bind to NMDA-Rs, but the physiological role of this interaction is not well understood. Here, we test if association between active CaMKII and synaptic NMDA-Rs is required for synaptic plasticity. Switching synaptic NR2B-containing NMDA-Rs that bind CaMKII with high affinity with those containing NR2A, a subunit with low affinity for CaMKII, dramatically reduces LTP. Expression of NR2A with mutations that increase association to active CaMKII recovers LTP. Finally, driving into synapses NR2B with mutations that reduce association to active CaMKII prevents LTP. Spontaneous activity-driven potentiation shows similar results. We conclude that association between active CaMKII and NR2B is required for different forms of synaptic enhancement. The switch from NR2B to NR2A content in synaptic NMDA-Rs normally observed in many brain regions may contribute to reduced plasticity by controlling the binding of active CaMKII.     

Urolithin A suppresses high glucose-induced neuronal amyloidogenesis by modulating TGM2-dependent ER-mitochondria contacts and calcium homeostasis

Hyperglycemia in diabetes mellitus (DM) patients is a causative factor for amyloidogenesis and induces neuropathological changes, such as impaired neuronal integrity, neurodegeneration, and cognitive impairment. Regulation of mitochondrial calcium influx from the endoplasmic reticulum (ER) is considered a promising strategy for the prevention of mitochondrial ROS (mtROS) accumulation that occurs in the Alzheimer’s disease (AD)-associated pathogenesis in DM patients. Among the metabolites of ellagitannins that are produced in the gut microbiome, urolithin A has received an increasing amount of attention as a novel candidate with anti-oxidative and neuroprotective effects in AD. Here, we investigated the effect of urolithin A on high glucose-induced amyloidogenesis caused by mitochondrial calcium dysregulation and mtROS accumulation resulting in neuronal degeneration. We also identified the mechanism related to mitochondria-associated ER membrane (MAM) formation. We found that urolithin A-lowered mitochondrial calcium influx significantly alleviated high glucose-induced mtROS accumulation and expression of amyloid beta (Aβ)-producing enzymes, such as amyloid precursor protein (APP) and β-secretase-1 (BACE1), as well as Aβ production. Urolithin A injections in a streptozotocin (STZ)-induced diabetic mouse model alleviated APP and BACE1 expressions, Tau phosphorylation, Aβ deposition, and cognitive impairment. In addition, high glucose stimulated MAM formation and transglutaminase type 2 (TGM2) expression. We first discovered that urolithin A significantly reduced high glucose-induced TGM2 expression. In addition, disruption of the AIP–AhR complex was involved in urolithin A-mediated suppression of high glucose-induced TGM2 expression. Markedly, TGM2 silencing inhibited inositol 1, 4, 5-trisphosphate receptor type 1 (IP3R1)–voltage-dependent anion-selective channel protein 1 (VDAC1) interactions and prevented high glucose-induced mitochondrial calcium influx and mtROS accumulation. We also found that urolithin A or TGM2 silencing prevented Aβ-induced mitochondrial calcium influx, mtROS accumulation, Tau phosphorylation, and cell death in neuronal cells. In conclusion, we suggest that urolithin A is a promising candidate for the development of therapies to prevent DM-associated AD pathogenesis by reducing TGM2-dependent MAM formation and maintaining mitochondrial calcium and ROS homeostasis.Subject terms: Cognitive ageing, Calcium and vitamin D     

不同寡聚形式的Aβ42与细胞膜作用显示毒性差异

β-淀粉样蛋白（β-amyloid peptide, Aβ）与神经细胞膜的相互作用是阿尔茨海默症（Alzheimer’s disease, AD）发病的重要事件,但不同寡聚形式的Aβ与细胞膜相互作用的差异仍缺乏直接比较。本文通过膜天平、透射电子显微镜、Thioflavin T(ThT)和细胞毒性实验等方法,检测Aβ42单体、ADDL、原纤维等形式的β-淀粉样蛋白与磷脂膜的作用方式,分析不同形式淀粉样蛋白对细胞的毒性作用。结果显示,（1）单层膜的实验数据可以判断Aβ42单体和寡聚体插膜能力存在差异,Aβ42单体能插入磷脂单层膜内,而Aβ42 ADDL不具备插膜能力;（2）透射电镜和ThT荧光检测,定性定量地分析出不同聚集形式的Aβ42具有不同的纤维化能力,Aβ42单体纤维化能力最强,而Aβ42原纤维的纤维化能力次之,Aβ42ADDL很难形成纤维;（3）Aβ42单体细胞毒性较弱,而Aβ42 ADDL和原纤维的细胞毒性较强。由以上结果可以得出结论：在磷脂膜存在的条件下,Aβ42单体可以插入膜内并迅速形成无毒性的Aβ42纤维,因此,细胞毒性较弱。而ADDL及原纤维不能插入膜内,纤维化能力较弱,从而以寡聚体的形式发挥细胞毒性。将单体、ADDL及原纤维形式的Aβ42与细胞膜相互作用进行分析,将为Aβ42在AD中的毒性机制研究提供一定的参考。但各种寡聚体入胞的方式及毒性机制仍需要进一步研究。     

APP processing and synaptic function

A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimer's disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.     

Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.     

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.     

Solution state characterization of amyloid beta-derived diffusible ligands

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.     

Astrocytes Express N-Methyl-D-Aspartate Receptor Subunits in Development,Ischemia and Post-Ischemia

The expression of the N-methyl-D-aspartate receptor (NMDA-R) in astrocytes is controversial. The receptor is commonly considered neuron-specific. We showed that astrocytes in primary cultures differentially expressed mRNA of NMDA-R subunits, NR1, NR2A and NR2B, in development, ischemia and post-ischemia. One-week-old cultures expressed detectable NR1 mRNA, which fell significantly at 2 weeks and became barely detectable at 4 weeks. NR2A and NR2B mRNA were both significantly up-regulated from 1 to 2 weeks. In 4 weeks, 2 h of ischemia caused a significant up-regulation of NR1 and NR2B mRNA; while 6 h caused down-regulation of NR2A mRNA. Under 3 h of post-ischemia, only NR1 mRNA was increased. Ischemia induced the expression of major NMDA-R effecter, nitric oxide synthase 1, which was unaffected by AMPA-R antagonist CNQX, but dose-dependently inhibited by NMDA-R specific antagonist MK-801. These findings reflected that astrocyte could express inducible functional NMDA receptors without the presence of neurons.     

Homocysteine potentiates β-amyloid neurotoxicity: role of oxidative stress

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The beta-amyloid fragment (Abeta) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Abeta neurotoxicity. HC potentiated the Abeta-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-L-cysteine blocked apoptosis following cotreatment with HC and Abeta, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Abeta.     

Mitochondrial permeability transition pore is a potential drug target for neurodegeneration

Mitochondrial permeability transition pore (mPTP) plays a central role in alterations of mitochondrial structure and function leading to neuronal injury relevant to aging and neurodegenerative diseases including Alzheimer's disease (AD). mPTP putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and cyclophilin D (CypD). Reactive oxygen species (ROS) increase intra-cellular calcium and enhance the formation of mPTP that leads to neuronal cell death in AD. CypD-dependent mPTP can play a crucial role in ischemia/reperfusion injury. The interaction of amyloid beta peptide (Aβ) with CypD potentiates mitochondrial and neuronal perturbation. This interaction triggers the formation of mPTP, resulting in decreased mitochondrial membrane potential, impaired mitochondrial respiration function, increased oxidative stress, release of cytochrome c, and impaired axonal mitochondrial transport. Thus, the CypD-dependent mPTP is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of AD. Designing small molecules to block this interaction would lessen the effects of Aβ neurotoxicity. This review summarizes the recent progress on mPTP and its potential therapeutic target for neurodegenerative diseases including AD. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca²⁺-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also μ-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, μ-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca²⁺ influx.