Impaired cellular copper regulation in the presence of ApoE4.

The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allelic variation of the APOE gene, with the following risk structure: ε4 > ε3 > ε2. The biochemical basis for this risk profile is unclear. Here, we reveal a new role for the APOE gene product, apolipoprotein E (ApoE) in regulating cellular copper homeostasis, which is perturbed in the AD brain. Exposure of ApoE target replacement (TR) astrocytes (immortalised astrocytes from APOE knock-in mice) to elevated copper concentrations resulted in exacerbated copper accumulation in ApoE4- compared to ApoE2- and ApoE3-TR astrocytes. This effect was also observed in SH-SY5Y neuroblastoma cells treated with conditioned medium from ApoE4-TR astrocytes. Increased intracellular copper levels in the presence of ApoE4 may be explained by reduced levels and delayed trafficking of the copper transport protein, copper-transporting ATPase 1 (ATP7A/Atp7a), potentially leading to impaired cellular copper export. This new role for ApoE in copper regulation lends further biochemical insight into how APOE genotype confers risk for AD and reveals a potential contribution of ApoE4 to the copper dysregulation that is a characteristic pathological feature of the AD brain.

Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress, Dysfunction of Calcium Signalling, and Inhibition of Autophagy-Lysosomal Pathway.

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-D-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic-lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.

Glutaredoxin1 protects neuronal cells from copper-induced toxicity.

Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.

Multifaceted role of nitric oxide in an in vitro mouse neuronal injury model: transcriptomic profiling defines the temporal recruitment of death signalling cascades.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

Transcriptional insights on the regenerative mechanics of axotomized neurons in vitro.

Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ± 1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.

Impaired insulin signaling in an animal model of Niemann-Pick Type C disease.

Studies have shown similarities between the histopathological characteristics of NPC and Alzheimer's disease (AD) including amyloid and tau pathologies. While dysfunction in insulin signaling was widely detected in AD brain, the function of insulin signaling proteins has not been examined in NPC disease. In this study, we have examined the expression and phosphorylation of proteins linked to the insulin signaling pathway in the brain of 9 weeks old NPC(nih) mice. Our results showed lower expression of insulin receptor substrate 2 (IRS2) in the NPC(nih) mice, and insulin receptor substrate 1 (IRS1) expression was almost non-detectable in this NPC mouse model. This reduction was associated with the loss of expression for the regulatory p85 subunit of phosphatidylinositol 3-kinase (p85/PI3K). Interestingly, the impairment was observed to link to a greater reduction of Akt phosphorylation at residue T308 than S473. This aberrant Akt phosphorylation could be contributing to lower GSK3ß phosphorylation detected in the NPC(nih) mouse brain. To our knowledge, this is the first report documenting impaired insulin signaling in the brain of a NPC mouse model.

The neuronal p35 activator of Cdk5 is a novel F-actin binding and bundling protein.

The neuronal Cdk5 activator p35 is involved in a multitude of neuronal activities, including cytoskeletal organization. We show here that p35 directly interacts with filamentous actin (F-actin) but not with monomeric actin (G-actin). Through binding, p35 induces the formation of actin bundles and stabilizes F-actin against dilution-induced depolymerization. p35 forms intermolecular self-associations, suggesting that p35 cross-links actin filaments into bundles via its intermolecular self-association. p35 dimerization and association with F-actin occur at the N-terminal region that is absent in the calpain-cleaved product p25, indicating that such p35 properties are lost by its truncation induced under neurotoxic conditions. Using p35 phosphorylated by Cdk5 and a mutational approach, we demonstrate that the phosphorylation of p35 promotes its homodimerization and p35-induced formation of F-actin bundles. In addition, the phosphorylation regulates p35 distribution to microtubule and actin cytoskeletons. Together, these observations define a novel function for p35 in cytoskeletal regulation.

Up-regulation of endoplasmic reticulum stress-related genes during the early phase of treatment of cultured cortical neurons by the proteasomal inhibitor lactacystin.

Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.

Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-D-aspartate receptor identifying commonalities with excitotoxicity.

Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.

Identification of p10 as a neurotoxic product generated from the proteolytic cleavage of the neuronal Cdk5 activator.

The involvement of cyclin-dependent kinase-5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron-fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent-resistant fraction made up of nuclear matrix, membrane-bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10-mediated apoptosis occurred via a caspases-independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent-insoluble fraction may be a crucial pathological event to triggering neuronal cell death.

Chlorinative stress: an under appreciated mediator of neurodegeneration?

Oxidative stress has been implicated as playing a role in neurodegenerative disorders, such as ischemic stroke, Alzheimer's, Huntington's, and Parkinson's disease. Persuasive evidences have shown that microglial-mediated oxidative stress contributes significantly to cell loss and accompanying cognitive decline characteristic of the diseases. Based on the facts that (i) levels of catalytically active myeloperoxidase are elevated in diseased brains and (ii) myeloperoxidase polymorphism is associated with the risk of developing neurodegenerative disorders, HOCl as a major oxidant produced by activated phagocytes in the presence of myeloperoxidase is therefore suggested to be involved in neurodegeneration. Its association with neurodegeneration is further showed by elevated level of 3-chlorotyrosine (bio-marker of HOCl in vivo) in affected brain regions as well as HOCl scavenging ability of neuroprotectants, desferrioxamine and uric acid. In this review, we will summary the current understanding concerning the association of HOCl and neuronal cell death where production of HOCl will lead to further formation of reactive nitrogen and oxygen species. In addition, HOCl also causes tissue destruction and cellular damage leading cell death.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways.

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint.

Antioxidants: promising neuroprotection against cardiotoxin-4b-induced cell death which triggers oxidative stress with early calpain activation.

Cardiotoxin-4b (CTX-4b), isolated from Naja naja sputatrix venom, shows lethality in several cell types. Employing murine primary cortical neurons, this study was undertaken to investigate the molecular mechanisms of CTX-4b in the induction of neuronal death. CTX-4b induced a dose- and time-dependent neuronal death. Strong induction of calpains as early as 4h post-CTX-4b 75 nM treatment was detected in neurons with negligible caspase 3 activation. For the first time in cultured murine primary cortical neurons, it was noted that CTX-4b-mediated cell death triggered oxidative stress with an increase in reactive oxygen species (ROS) levels, and that application of antioxidants showed effective attenuation of cell death. Taken together, these results indicate that CTX-4b-mediated neuronal death is associated with (i) early calpain activation and (ii) oxidative stress. Most importantly, antioxidants have proved to be a promising therapeutic avenue against CTX-4b-induced neuronal death.

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk.

Excitotoxicity mediated via the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l-glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (deltapsi(m)), early release of cytochrome c (cyt c), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of deltapsi(m), and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke feltapsi(m) as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c, SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.

Hydrogen sulfide induced neuronal death occurs via glutamate receptor and is associated with calpain activation and lysosomal rupture in mouse primary cortical neurons.

Hydrogen sulfide (H(2)S) is a cytotoxic gas recently proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM and perturbed H(2)S synthesis has been reported in the brains from stroke, Alzheimer's disease and Down syndrome patients. Recently, in immature non-glutamate receptor expressing mouse cortical neurons H(2)S was shown to inhibit cell death exhibited by high concentrations of glutamate whereas H(2)S was not cytotoxic. Due to the reported role of H(2)S in facilitating LTP through NMDA receptors we examined the effects of H(2)S on glutamate receptor functioning using mature cortical neurons expressing functional glutamate receptor subtypes. Addition of 100 microM glutamate exhibited extensive cell death which was exacerbated by co-incubation with < or = 200 microM of the H(2)S donor sodium hydrosulfide (NaHS). At <200 microM NaHS induced apoptosis whereas >200 microM NaHS induced necrosis. Cell death was inhibited by pharmacological glutamate receptor antagonists MK801 and APV (NMDA receptor antagonists), and CNQX (kainate and AMPA receptor antagonist) but not kynurenate (broad spectrum glutamate receptor antagonist), GYKI52466 (more selective AMPA receptor antagonist) and CYZ (AMPA receptor potentiator). Although markers of apoptosis were observed, we did not detect caspase activation either by Western blotting or fluorescence assays and caspase inhibitors did not prevent cell death. Rather, H(2)S induced calpain activation and lysosomal membrane destabilization; processes inhibited by preferential antagonists of NMDA and kainate receptors. These data suggest that H(2)S induced neuronal death through ionotropic glutamate receptors, which recruits apoptosis to ensure cellular demise and employs calpains and lysosomal rupture. This study provides novel insights into cell death observed in neurodegenerative diseases involving glutamate receptor activation and perturbed H(2)S synthesis.

Deciphering the mechanism of HNE-induced apoptosis in cultured murine cortical neurons: transcriptional responses and cellular pathways.

Studies have shown that the lipid peroxidation by-product, 4-hydroxynonenal (HNE), is involved in many pathological events in several neurodegenerative diseases. A number of signaling pathways mediating HNE-induced cell death in the brain have been proposed. However, the exact mechanism remains unknown. In the present study, we have examined the effects of HNE on cultured primary cortical neurons and found that HNE treatment leads to cell death via apoptosis. Both the caspase and calpain proteolytic systems were activated. There were also increased levels of phospho-p53 and cell cycle-related proteins. Gene transcription was further studied using microarray analysis. Results showed that majority of the genes associated with cell cycle regulation, response to stress, and signal transduction were differentially expressed. The various categories of differentially-expressed genes suggested that there are other parallel pathways regulating HNE-induced neuronal apoptosis. Collectively, these might help to elucidate similar molecular mechanisms involved during cell death in neurodegenerative diseases.

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model.

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.

Cellular mechanism of U18666A-mediated apoptosis in cultured murine cortical neurons: bridging Niemann-Pick disease type C and Alzheimer's disease.

Neuronal cell death can occur by means of either necrosis or apoptosis. Both necrosis and apoptosis are generally believed to be distinct mechanisms of cell death with different characteristic features distinguished on the basis of their morphological and biochemical properties. The brain is the most cholesterol-rich organ in the body but not much is known about the mechanisms that regulate cholesterol homeostasis in the brain. Recently, several clinical and biochemical studies suggest that cholesterol imbalance in the brain may be a risk factor related to the development of neurological disorders such as Niemann-Pick disease type C (NPC) and Alzheimer's disease (AD). NPC is a fatal juvenile neurodegenerative disorder characterized by premature neuronal death and somatically altered cholesterol metabolism. The main biochemical manifestation in NPC is elevated intracellular accumulation of free cholesterol caused by a genetic deficit in cholesterol trafficking. The pharmacological agent, U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), is a well-known class-2 amphiphile which inhibits cholesterol transport. Cells treated with this agent accumulate intracellular cholesterol to massive levels, similar to that observed in cells from NPC patients. NPC and AD have some pathological similarities which may share a common underlying cause. AD is one of the most common types of dementia affecting the elderly. However, the molecular mechanisms of neurodegeneration in NPC and AD are largely unknown. This review provides a consolidation of work done using U18666A in the past half century and focuses on the implications of our research findings on the mechanism of U18666A-mediated neuronal apoptosis in primary cortical neurons, which may provide an insight to elucidate the mechanisms of neurodegenerative diseases, particularly NPC and AD, where apoptosis might occur through a similar mechanism.

U18666A-mediated apoptosis in cultured murine cortical neurons: role of caspases, calpains and kinases.

Studies have suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders such as Alzheimer's disease and Niemann-Pick disease type C. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we examined the effects of U18666A-mediated neuronal apoptosis, and found that chronic exposure to U18666A led to the activation of caspases and calpains and hyperphosphorylation of tau. Tau hyperphosphorylation is regulated by several kinases that phosphorylate specific sites of tau in vitro. Surprisingly, the kinase activity of cyclin-dependent kinase 5 decreased in U18666A-treated cortical neurons whereas its protein level remained unchanged. The amount of glycogen synthase kinase 3 and mitogen-activated protein kinases were found to decrease in their phosphorylated states by Western blot analysis. Gene transcription was further studied using microarray analysis. Genes encoding for kinases and phosphatases were differentially expressed with most up-regulated and some down-regulated in expression upon U18666A treatment. The activation of cysteine proteases and cholesterol accumulation with tauopathies may provide clues to the cellular mechanism of the inhibition of cholesterol transport-mediated cell death in neurodegenerative diseases.

The very C-terminus of protein kinase Cepsilon is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1.

In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.