Trophic Withdrawal Research Articles

P75NTR and DR6 Regulate Distinct Phases of Axon Degeneration Demarcated by Spheroid Rupture.

The regressive events associated with trophic deprivation are critical for sculpting a functional nervous system. After nerve growth factor withdrawal, sympathetic axons derived from male and female neonatal mice maintain their structural integrity for ∼18 h (latent phase) followed by a rapid and near unison disassembly of axons over the next 3 h (catastrophic phase). Here we examine the molecular basis by which axons transition from latent to catastrophic phases of degeneration following trophic withdrawal. Before catastrophic degeneration, we observed an increase in intra-axonal calcium. This calcium flux is accompanied by p75 neurotrophic factor receptor-Rho-actin-dependent expansion of calcium-rich axonal spheroids that eventually rupture, releasing their contents to the extracellular space. Conditioned media derived from degenerating axons are capable of hastening transition into the catastrophic phase of degeneration. We also found that death receptor 6, but not p75 neurotrophic factor receptor, is required for transition into the catastrophic phase in response to conditioned media but not for the intra-axonal calcium flux, spheroid formation, or rupture that occur toward the end of latency. Our results support the existence of an interaxonal degenerative signal that promotes catastrophic degeneration among trophically deprived axons.SIGNIFICANCE STATEMENT Developmental pruning shares several morphological similarities to both disease- and injury-induced degeneration, including spheroid formation. The function and underlying mechanisms governing axonal spheroid formation, however, remain unclear. In this study, we report that axons coordinate each other's degeneration during development via axonal spheroid rupture. Before irreversible breakdown of the axon in response to trophic withdrawal, p75 neurotrophic factor receptor-RhoA signaling governs the formation and growth of spheroids. These spheroids then rupture, allowing exchange of contents ≤10 kDa between the intracellular and extracellular space to drive death receptor 6 and calpain-dependent catastrophic degeneration. This finding informs not only our understanding of regressive events during development but may also provide a rationale for designing new treatments toward myriad neurodegenerative disorders.

Axonal Degeneration Is Regulated by a Transcriptional Program that Coordinates Expression of Pro- and Anti-degenerative Factors

Developmental neuronal cell death and axonal elimination are controlled by transcriptional programs, of which their nature and the function of their components remain elusive. Here, we identified the dual specificity phosphatase Dusp16 as part of trophic deprivation-induced transcriptome in sensory neurons. Ablation of Dusp16 enhanced axonal degeneration in response to trophic withdrawal, suggesting that it has a protective function. Moreover, axonal skin innervation was severely reduced while neuronal elimination was increased in the Dusp16 knockout. Mechanistically, Dusp16 negatively regulates the transcription factor p53 and antagonizes the expression of the pro-degenerative factor, Puma (p53 upregulated modulator of apoptosis). Co-ablation of Puma with Dusp16 protected axons from rapid degeneration and specifically reversed axonal innervation loss early in development with no effect on neuronal deficits. Overall, these results reveal that physiological axonal elimination is regulated by a transcriptional program that integrates regressive and progressive elements and identify Dusp16 as a new axonal preserving factor.

Over-expression of the Sirt3 sirtuin Protects neuronally differentiated PC12 Cells from degeneration induced by oxidative stress and trophic withdrawal

Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45kDa pro-form, which localized primarily within the cytosol, and a 30kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen–glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.

Sarm1-Mediated Axon Degeneration Requires Both SAM and TIR Interactions

Axon degeneration is an evolutionarily conserved pathway that eliminates damaged or unneeded axons. Manipulation of this poorly understood pathway may allow treatment of a wide range of neurological disorders. In an RNAi-based screen performed in cultured mouse DRG neurons, we observed strong suppression of injury-induced axon degeneration upon knockdown of Sarm1 [SARM (sterile α-motif-containing and armadillo-motif containing protein)]. We find that a SARM-dependent degeneration program is engaged by disparate neuronal insults: SARM ablation blocks axon degeneration induced by axotomy or vincristine treatment, while SARM acts in parallel with a soma-derived caspase-dependent pathway following trophic withdrawal. SARM is a multidomain protein that associates with neuronal mitochondria. Deletion of the N-terminal mitochondrial localization sequence disrupts SARM mitochondrial localization in neurons but does not alter its ability to promote axon degeneration. In contrast, mutation of either the SAM (sterile α motif) or TIR (Toll-interleukin-1 receptor) domains abolishes the ability of SARM to promote axonal degeneration, while a SARM mutant containing only these domains elicits axon degeneration and nonapoptotic neuronal death even in the absence of injury. Protein-protein interaction studies demonstrate that the SAM domains are necessary and sufficient to mediate SARM-SARM binding. SARM mutants lacking a TIR domain bind full-length SARM and exhibit strong dominant-negative activity. These results indicate that SARM plays an integral role in the dismantling of injured axons and support a model in which SAM-mediated multimerization is necessary for TIR-dependent engagement of a downstream destruction pathway. These findings suggest that inhibitors of SAM and TIR interactions represent therapeutic candidates for blocking pathological axon loss and neuronal cell death.

Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal

The goal of this study was to compare the neuroprotective properties of the l-type Ca2+ channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)-differentiated PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) and trophic withdrawal-induced cell death. Nimodipine (1–100μM) conferred 65±13% neuroprotection upon exposure to OGD and 35±6% neuroprotection towards different trophic withdrawal-induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5h but not at longer OGD exposures. Nifedipine (1–100μM), to a lower potency than nimodipine, conferred 30–55±8% neuroprotection towards OGD in PC12 cells and 29±5% in rat hypocampal slices, and 10±3% neuroprotection at 100μM towards trophic withdrawal-induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time-window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.

Image-based Screening Identifies Novel Roles for IκB Kinase and Glycogen Synthase Kinase 3 in Axonal Degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.

Myocardin-related transcription factor-A promoting neuronal survival against apoptosis induced by hypoxia/ischemia

MRTF-A, known as one of the myocardin-related transcription factors, is widely found in newborn rat cortical or hippocampus neurons as well as in adult rat forebrain. Recent studies indicate that MRTF-A elevates SRF-driven transcription and enhances its stimulation by neurotrophic factor (BDNF). However, the mechanism underlying the contribution of the MRTF-A to neuronal survival is not completely understood. In this study, we investigated the effect of MRTF-A on neuronal apoptosis and its underlying mechanism. First of all, our study demonstrated that MRTF-A expression decreased obviously in rats' brains during the early period of cerebral ischemia–reperfusion. In order to estimate the effect of MRTF-A on neuronal apoptosis in vitro, we used an established experimental paradigm in which MRTF-A protected cortical neurons against both hypoxia–trophic deprivation and hydrogen peroxide-induced apoptosis. Obviously, over-expression of wild-type MRTF-A in cortical neurons inhibited apoptosis rate and enhanced anti-apoptotic gene-MCL-1. In contrast, co-expression of MRTF-A and the small interfering RNA of MRTF-A (siRNA) reversed the effect of neuroprotection and the upregulation on MCL-1 expression afforded by MRTF-A. Our study also determined whether the effect of MRTF-A up-regulating on MCL-1 expression is correlated to MRTF-A-enhancing CArG box transcription. The result showed that over-expression of wild-type MRTF-A upregulated the transcription activity of MCL reporter gene via driving the binding-domain CArG box in MCL-1 promoter, which was also reversed by co-expression of MRTF-A siRNA. In addition, we also found that BDNF neuroprotection on apoptosis induced by hypoxia–trophic withdrawal was not only inhibited by LY29004 and PD98059 but also partially blocked by transfection of dominant-negative MRTF-A in cortical neurons via enhancing the expression of anti-apoptotic gene-MCL-1, suggesting a downstream neuroprotective mechanism to BDNF neuroprotection on apoptosis.

Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-beta peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-beta-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.

Phosphorylation of Neuronal Survival Factor MEF2D by Glycogen Synthase Kinase 3β in Neuronal Apoptosis

Glycogen synthase kinase 3beta (GSK3beta) has been identified to play important roles in neuronal death. Evidence from both in vitro and in vivo studies indicates that increased GSK3beta activity contributes to neurodegeneration and to the pathogenesis of Alzheimer disease. But the molecular mechanisms that underlie GSK3beta-mediated neurotoxicity remain poorly understood. We reported here that myocyte enhancer factor 2D (MEF2D), a nuclear transcription factor known to promote neuronal survival, is directly phosphorylated by GSK3beta. Our data showed that phosphorylation of MEF2D by GSK3beta at three specific residues in its transactivation domain inhibits MEF2D transcriptional activity. Withdrawal of neuronal activity in cerebellar granule neurons activated GSK3beta in the nucleus, leading to GSK3beta-dependent inhibition of MEF2 function. This inhibition contributed to GSK3beta-mediated neuronal toxicity. Overexpression of MEF2D mutant that is resistant to GSK3beta inhibition protected cerebellar granule neurons from either GSK3beta activation- or neuronal activity deprivation-induced toxicity. These results identify survival factor MEF2D as a novel downstream effector targeted by GSK3beta and define a molecular link between activation of GSK3beta and neuronal survival machinery which may underlie in part GSK3beta-mediated neurotoxicity.

Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro

Bone marrow-derived mesenchymal stem cells (MSCs) are of therapeutic interest in a variety of neurological diseases. In this study, we wished to determine whether human MSCs secrete factors which protect cultured rodent cortical neurons from death by trophic factor withdrawal or nitric oxide (NO) exposure. Medium conditioned by MSCs attenuated neuronal death under these conditions, a process which was dependent on intact PI 3kinase/Akt pathway signaling. Trophic withdrawal and NO exposure in cultured cortical neurons led to reduction in Akt signaling pathways, whereas NO administration activated p38 MAPkinase in neuronal cultures. Addition of MSC-conditioned medium significantly activated the PI3kinase/Akt pathway and in neurons exposed to NO, MSC-conditioned medium reduced p38 signaling. We show that MSCs secrete brain-derived neurotrophic factor (BDNF) and addition of anti-BDNF neutralising antibodies to MSC-conditioned medium attenuated its neuroprotective effect. Exposure of neurons to BDNF increased activation of Akt pathways and protected neurons from trophic factor withdrawal. These observations determine the mechanisms of neuroprotection offered by MSC-derived factors and suggest an important role for BDNF in neuronal protection.

The expression of tumor necrosis factor-alpha, its receptors and steroidogenic acute regulatory protein during corpus luteum regression

BackgroundCorpus luteum (CL) regression is known to occur as two parts; functional regression when steroidogenesis declines and structural regression when apoptosis is induced. Previous studies suggest this process occurs by the production of luteolytic factors, such as tumour necrosis factor-alpha (TNF-alpha).MethodsWe examined TNF-alpha, TNF-alpha receptors (TNFR1 and 2) and steroidogenic acute regulatory (StAR) protein expression during CL regression in albino Wistar rats. CL from Days 16 and 22 of pregnancy and Day 3 post-partum were examined, in addition CL from Day 16 of pregnancy were cultured in vitro to induce apoptosis. mRNA was quantitated by kinetic RT-PCR and protein expression examined by immunohistochemistry and Western blot analyses.ResultsTNF-alpha mRNA increased on Day 3 post-partum. TNFR were immunolocalized to luteal cells, and an increase in TNFR2 mRNA observed on Day 3 post-partum whilst no change was detected in TNFR1 mRNA relative to Day 16. StAR protein decreased on Day 3 post-partum and following trophic withdrawal but no change was observed following exogenous TNF-alpha treatment. StAR mRNA decreased on Day 3 post-partum; however, it increased following trophic withdrawal and TNF-alpha treatment in vitro.ConclusionThese results demonstrate the existence of TNFR1 and TNFR2 in rat CL and suggest the involvement of TNF-alpha in rat CL regression following parturition. Furthermore, decreased StAR expression over the same time points was consistent with the functional regression of the CL.

Human neuroblastoma cells transfected with two Chinese presenilin 1 mutations are sensitized to trophic factor withdrawal and protected by insulin-like growth factor-1

Two novel presenilin 1 (PS1) mutations, V97L and A136G, were recently found to be involved in the early-onset of Alzheimer's disease in two Chinese families. This research aimed to verify their pathological effects. The human neuroblastoma SH-SY5Y cells stably transfected with these two Chinese presenilin 1 mutations were established to explore whether they are sensitive to, or influenced by, serum deprivation and protected by insulin-like growth factor-1 (IGF-1). Apoptosis rate, glucose uptake of the cells and the expression of glucose transport protein 1 (GLUT1) on cell membranes were examined. The V97L or A136G mutants significantly decreased the cells viability and increased the apoptosis rate when compare to PS1wt and mock transfected cells. IGF-1 was found to improve the viability of these two kinds of mutant cells significantly, and to show a protective effect for the mutants when they were treated with trophic deprivation. The glucose uptake of each transfected cell line increased to about 25% after IGF-1 treatment, GLUT1 expression on the cell membrane increased modestly by about 15% - 20%. Enhanced sensitivity to trophic withdrawal in the cells transfected with the two Chinese PS1 mutations may contribute to the neuron apoptosis. IGF-1 provided a protective effect to cells, possibly through an enhanced glucose transport and mitochondrial activities.

Levels of protein tyrosine phosphatase 1B determine susceptibility to apoptosis in serum‐deprived hepatocytes

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. To assess the importance of PTP1B in the balance between death and survival in the liver, we have developed immortalized neonatal hepatocyte cell lines lacking (PTP1B(-/-)) or overexpressing (PTP1B(+/+PTP1B)) PTP1B. Early activation of caspase-3 occurred in PTP1B(+/+PTP1B) hepatocytes but was nearly abolished in PTP1B(-/-) cells. At the molecular level, PTP1B overexpression/deficiency altered the balance of pro-(Bim) and anti-(Bcl-x(L)) apoptotic members of the Bcl-2 family upon serum withdrawal. Likewise, cytosolic cytochrome C increased rapidly in PTP1B(+/+PTP1B) hepatocytes whereas it was retained in the mitochondria of PTP1B(-/-) cells. DNA fragmentation and the increase of apoptotic cells induced by serum withdrawal in wild-type (PTP1B(+/+)) hepatocytes were absent in PTP1B(-/-) cells. Conversely, overexpression of PTP1B accelerated DNA laddering and increased the number of apoptotic cells. In serum-deprived PTP1B(+/+PTP1B) hepatocytes, a rapid entry of Foxo1 into the nucleus and an earlier activation of caspase-8 was observed. However, both events were suppressed in PTP1B(-/-) hepatocytes. Moreover, PTP1B deficiency conferred resistance to apoptosis induced by activation of Fas and constitutively active Foxo1. Rescue of PTP 1B in deficient hepatocytes recovered the phenotype of wild-type cells whereas reduction of PTP1B by siRNA suppressed apoptosis. Our results reveal a unique role for PTP1B as a mediator of the apoptotic pathways triggered by trophic factors withdrawal in hepatocytes. This novel mechanism may represent an important target in the design of therapeutic strategies for human liver regeneration after pathological damage as well as for treatment of hepatocarcinomas.

The effect of two newly Chinese presenilin-1 mutations on the sensitivity to trophic factor withdrawal in human neuroblastoma cells

To study the effects of two novel presenilin-1 (PS1) mutations, V97L and A136G, discovered in 2 families of Alzheimer's disease (AD) in China, on human neuroblastoma cells and the protective role of insulin-like growth factor-1 (IGF-1). Two mutation type (MT) PS-1 genes with the mutational sites V97L and A136G were established. These 2 MT genes, wild type (WT) PS-1 gene, and mock were stably transfected into the human neuroblastoma cells of the line SH-SY5Y. The 4 kinds of transfected cells were divided into 3 subgroups: (1) control subgroup, cultured in normal medium with serum, (2) serum deprivation subgroup, cultured in the medium without serum, and (3) serum deprivation + IGF-1 subgroup, cultured in serum deprivation medium plus IGF-1. Twenty-four hours later MTT colorimetry was used to calculate the cell survival rate. Hoechst33258 method and phosphatidylinositol/annexin V-FITC double staining flow cytometry were used to observe the cell apoptosis. The glucose uptake was measured. Western blotting was used to analyze the protein expression of glucose transporter protein (GLUT)-1 in the cell membrane. Different tests all showed that the cell survival rates of the V97L and A136G serum deprivation subgroups were both significantly lower than those of the corresponding WT and Mock subgroups (P < 0.01, P < 0.05), the cell survival rates of the all serum deprivation + IGF-1 subgroups were significantly higher than those of the corresponding serum deprivation subgroups (all P < 0.01); The glucose uptake rate was not significantly different among different subgroups, but IGF-1 addition made the glucose uptake increase by about 25% (all P > 0.05). The GLUT1 expression in the cell membrane of the serum deprivation + IGF-I subgroups increased by 15% - 20% respectively, however, without significant differences. Enhanced sensitivity to trophic withdrawal in the cells with the two Chinese PS1 mutations most likely contributes to the neuron loss in AD. The sensitivity to apoptosis is not caused by impaired glucose metabolism, but IGF-1 still can protect cells from serum deprivation that compensates the negative effect through enhancing glucose transport and mitochondria activities.

Expression of an Uncleavable N-terminal RasGAP Fragment in Insulin-secreting Cells Increases Their Resistance toward Apoptotic Stimuli without Affecting Their Glucose-induced Insulin Secretion

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.

DHA Increases PS to Promote Neuronal Survival

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid, and various neural diseases and deficits in development of the nervous system are associated with deficiency in this lipid. DHA is a precursor for phosphatidylserine (PS) biosynthesis, and addition of DHA increases PS content in brain or in cultured glial or neuronal cells. Akbar et al. show that supplementation of Neuro 2A cells with varying ratios of DHA and DPA (docosapentaenoic acid, a saturated lipid) showed that DHA promoted the greatest increase in PS concentration and was most effective at preventing apoptosis in cells deprived of serum. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) blocked the cell survival effect of DHA, and enrichment of cells with DHA partially rescued the phosphorylation and activity of the protein kinase Akt (a downstream effector in the PI3K pathway) in the absence of serum. Neuro 2A cells enriched with DHA or DPA (both of which increase PS concentration) showed faster recruitment of Akt to the membrane and faster phosphorylation of Akt upon stimulation of the cells with insulin-like growth factor (IGF). DHA resulted in the fastest redistribution, and this faster redistribution was not seen if the cells were grown in serine-depleted medium, suggesting that the increase in PS was responsible for this effect. In support of Akt directly interacting with PS, mutant forms of Akt (R67A or R69A) that may not bind acidic phospholipids translocated to a lesser extent to the membrane upon cell stimulation, and DHA or DPA did not alter the translocation of these mutants to the membrane. Hippocampal neurons from embryonic rats isolated from a rat fed either a normal or an n-3 fatty acid-deficient diet showed that the cells from the deficient rats had 15% less PS and that the cells were more susceptible to cell death following trophic withdrawal than were the DHA-replete cells. Thus, the abundance of PS in the inner plasma membrane leaflet appears to contribute to the recruitment of Akt in the cell survival pathway in neurons, and this may explain the neuronal deficits associated with n-3 fatty acid deficiency. M. Akbar, F. Calderon, Z. Wen, H. Y. Kim, Docosahexaenoic acid: A positive modulator of Akt signaling in neuronal survival. Proc. Natl. Acad. Sci. U.S.A. 102 , 10858-10863 (2005). [Abstract] [Full Text]

Neuroprotective effects of novel small peptides in vitro and after brain injury

Thyrotropin-releasing hormone (TRH) and TRH analogues have been reported to be neuroprotective in experimental models of spinal cord injury and head injury. We have previously shown that a diketopiperazine structurally related to the TRH metabolite cyclo-his-pro reduces neuronal cell death in vitro and in vivo. Here we report the neuroprotective activity of other cyclic dipeptides in multiple in vitro models of neuronal injury and after controlled cortical impact (CCI) in mice. Using primary neuronal cultures, three novel dipeptides were compared to the previously reported diketopiperazine as well as to vehicle controls; each of the compounds reduced cell death after direct physical trauma or trophic withdrawal. Two of these peptides also protected against glutamate toxicity and β-amyloid-induced injury; the latter also strongly inhibited glutamate-induced increases in intracellular calcium. Treatment with each of the test compounds resulted in highly significant improvement of motor and cognitive recovery after CCI, as well as markedly reducing lesion volumes as shown by high field magnetic resonance imaging. DNA microarray studies following fluid percussion induced traumatic brain injury (TBI) in rats showed that treatment with one of these dipeptides after injury significantly down-regulated expression of mRNAs for cell cycle proteins, aquaporins, cathepsins and calpain in ipsilateral cortex and/or hippocampus, while up-regulating expression of brain-derived neurotrophic factor, hypoxia-inducible factor and several heat-shock proteins. Many of these mRNA expression changes were paralleled at the protein level. The fact that these small peptides modulate multiple mechanisms favoring neuronal cell survival, as well as their ability to improve functional outcome and reduce posttraumatic lesion size, suggests that they may have potential utility in clinical head injury.

Novel small peptides with neuroprotective and nootropic properties

The tripeptide thyrotropin-releasing hormone (TRH) and/or related analogues have shown neuroprotective activity across multiple animal trauma models as well as in a small clinical trial of spinal cord injury. The metabolic product of TRH (cyclo-his-pro) retains physiological activity. We have developed a number of novel cyclic dipeptides that are structurally similar to cyclo-his-pro, and have examined their neuroprotective activity across multiple in vitro models of neuronal injury and after traumatic brain injury (TBI) in rodents. Four such compounds were found to reduce cell death after trophic withdrawal or traumatic injury in primary neuronal cultures; two also protected against glutamate or beta-amyloid neurotoxicity. All compounds significantly improved motor and cognitive recovery after controlled cortical impact injury in mice, and markedly reduced lesion volumes as shown by high field magnetic resonance imaging. Further, compound 35b, which is being developed for clinical trials, also showed considerable neuroprotection after fluid percussion induced TBI in rats, and improved cognitive function after daily administration in chronically brain injured rats. At a mechanistic level, the drugs attenuate both apoptotic and necrotic cell death in primary neuronal cultures, markedly reduce intracellular calcium accumulation after injury, and limit changes in mitochondrial membrane potential and associated cytochrome c release. In addition, microarray studies show that 35b reduces transcriptional changes after injury for a number of genes (and proteins) that may be associated with secondary injury, including cell cycle genes, aquaporins and cathepsins. It also upregulates brain-derived neurotrophic factor (BDNF), heat shock proteins (HSP) and hypoxia inducible factor (HIF). Thus, these novel dipeptides have multipotential actions that make them candidates for the treatment of both acute and chronic neurodegeneration.

JNK2 Translocates to the Mitochondria and Mediates Cytochrome c Release in PC12 Cells in Response to 6-Hydroxydopamine

6-Hydroxydopamine (6-OHDA) causes death of dopaminergic neurons by mitochondrial dysfunction with JNKs as central mediators. Here we provide novel insights into specific actions of JNK isoforms in 6-OHDA-induced death of PC12 cells. Twenty five mum 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus, and at the mitochondria. Inhibition of JNKs by 2 mum SP600125 or transfection with dominant-negative JNK2 (dnJNK2) rescued more than 60% of the otherwise dying PC12 cells after 24 h, whereas transfection with dnJNK1 had no protective effects. In contrast to constitutively present JNK1, JNK2 amounts increased in the nucleus and at the mitochondria after 6-OHDA stimulation. JNK inhibition by SP600125 or transfection of dnJNK2 reduced the pool of active JNKs in the nucleus, the release of cytochrome c, as well as the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase-1. Transfection with dnJNK1, however, had no effects on the translocation of JNKs to the mitochondria or the release of cytochrome c. Our data provide novel functional insights into the pathological role of individual JNK isoforms, the signalosome at the mitochondria, and the mode of JNK-induced release of cytochrome c.