Search

Motivation: The collective and co-ordinated synaptic activity of large neuronal populations is relevant to neuronal development as well as a range of neurological diseases. Quantification of synaptically-mediated neuronal signalling permits further downstream analysis as well as potential application in target validation and in vitro screening assays. Our aim is to develop a phenotypic quantification for neuronal activity imaging data of large populations of neurons, in particular relating to the spatial component of the activity., Results: We extend the use of Markov random field (MRF) models to achieve this aim. In particular, we consider Bayesian posterior densities of model parameters in Gaussian MRFs to directly model changes in calcium fluorescence intensity rather than using spike trains. The basis of our model is defining neuron 'neighbours' by the relative spatial positions of the neuronal somata as obtained from the image data whereas previously this has been limited to defining an artificial square grid across the field of view and spike binning. We demonstrate that our spatial phenotypic quantification is applicable for both in vitro and in vivo data consisting of thousands of neurons over hundreds of time points. We show how our approach provides insight beyond that attained by conventional spike counting and discuss how it could be used to facilitate screening assays for modifiers of disease-associated defects of communication between cells., Availability and Implementation: We supply the MATLAB code and data to obtain all of the results in the paper., Supplementary Information: Supplementary data are available at Bioinformatics online.

Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.

The protein NOS1AP/CAPON mediates signaling from a protein complex of NMDA receptor, PSD95 and nNOS. The only stroke trial for neuroprotectants that showed benefit to patients targeted this ternary complex. NOS1AP/nNOS interaction regulates small GTPases, iron transport, p38MAPK-linked excitotoxicity, and anxiety. Moreover, the nos1ap gene is linked to disorders from schizophrenia, post-traumatic stress disorder, and autism to cardiovascular disorders and breast cancer. Understanding protein interactions required for NOS1AP function, therefore, has broad implications for numerous diseases. Here we show that the interaction of NOS1AP with nNOS differs radically from the classical PDZ docking assumed to be responsible. The NOS1AP PDZ motif does not bind nNOS as measured by multiple methods. In contrast, full-length NOS1AP forms an unusually stable interaction with nNOS. We mapped the discrepancy between full-length and C-terminal PDZ motif to a novel internal region we call the ExF motif. The C-terminal PDZ motif, although neither sufficient nor necessary for binding, nevertheless promotes the stability of the complex. It therefore potentially affects signal transduction and suggests that functional interaction of nNOS with NOS1AP might be targetable at two distinct sites. We demonstrate that excitotoxic pathways can be regulated, in cortical neuron and organotypic hippocampal slice cultures from rat, either by the previously described PDZ ligand TAT-GESV or by the ExF motif-bearing region of NOS1AP, even when lacking the critical PDZ residues as long as the ExF motif is intact and not mutated. This previously unrecognized heterodivalent interaction of nNOS with NOS1AP may therefore provide distinct opportunities for pharmacological intervention in NOS1AP-dependent signaling and excitotoxicity., (Copyright © 2015 the authors 0270-6474/15/357349-16$15.00/0.)

Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neurodegenerative conditions, but the intermediary mechanism is unclear. NOS1AP is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity.

Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

Cell migration is the consequence of the sum of positive and negative regulatory mechanisms. Although appropriate migration of neurons is a principal feature of brain development, the negative regulatory mechanisms remain obscure. We found that JNK1 was highly active in developing cortex and that selective inhibition of JNK in the cytoplasm markedly increased both the frequency of exit from the multipolar stage and radial migration rate and ultimately led to an ill-defined cellular organization. Moreover, regulation of multipolar-stage exit and radial migration in Jnk1(-/-) (also known as Mapk8) mice, resulted from consequential changes in phosphorylation of the microtubule regulator SCG10 (also called stathmin-2). Expression of an SCG10 mutant that mimics the JNK1-phosphorylated form restored normal migration in the brains of Jnk1(-/-) mouse embryos. These findings indicate that the phosphorylation of SCG10 by JNK1 is a fundamental mechanism that governs the transition from the multipolar stage and the rate of neuronal cell movement during cortical development.

JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective.

Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3beta, but not GSK-3alpha, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and regulation of Bim protein turnover despite constitutive cycling of GSK-3 between the cytosol and nucleus, revealed by leptomycin B. We examine the importance of Ser21/9 (GSK-3alpha/beta) phosphorylation on proapoptotic GSK-3 function. Neurons isolated from GSK-3alpha/beta(S21A/S9A) knock-in mice survive normally and are fully sensitive to trophic-deprivation-induced death. Nonetheless, inhibition of GSK-3 catalytic activity with lithium or SB216763 protects GSK-3alpha/beta(S21A/S9A) neurons from death. This indicates that dephosphorylation of GSK-3beta/Ser9 and GSK-3alpha/Ser21 is insufficient for GSK-3 proapoptotic function and that another level of regulation is required. Gel filtration reveals a stress-induced loss of neuronal GSK-3beta from a high-molecular-mass complex with a concomitant decrease in axin-bound GSK-3beta. These data imply that Wnt-regulated GSK-3beta plays a nonredundant role in trophic-deprivation-induced death of neurons.

Excitotoxic neuronal death contributes to many neurological disorders, and involves calcium influx and stress-activated protein kinases (SAPKs) such as p38alpha. There is indirect evidence that the small Rho-family GTPases Rac and cdc42 are involved in neuronal death subsequent to the withdrawal of nerve growth factor (NGF), whereas Rho is involved in the inhibition of neurite regeneration and the release of the amyloidogenic Abeta(42) peptide. Here we show that Rho is activated in rat neurons by conditions that elevate intracellular calcium and in the mouse cerebral cortex during ischemia. Rho is required for the rapid glutamate-induced activation of p38alpha and ensuing neuronal death. The ability of RhoA to activate p38alpha was not expected, and it was specific to primary neuronal cultures. The expression of active RhoA alone not only activated p38alpha but also induced neuronal death that was sensitive to the anti-apoptotic protein Bcl-2, showing that RhoA was sufficient to induce the excitotoxic pathway. Therefore, Rho is an essential component of the excitotoxic cell death pathway.

The stress-activated protein kinases c-Jun-activated kinase (JNK) and p38 are implicated in neuronal apoptosis. Early studies in cell lines suggested a requirement for both in the apoptosis induced by withdrawal of nerve growth factor. However, studies in neuronal cells typically implicate JNK but not p38 in apoptosis. In some cases, p38 is implicated, but the role of JNK is undefined. It remains unclear whether p38 and JNK have differing roles dependent on cell type, apoptotic stimulus, or mechanism of cell death or whether they are redundant and each sufficient to induce identical forms of cell death. We investigate the relative roles of these protein kinases in different death mechanisms in a single system, cultured cerebellar granule neurons. Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different in terms of caspase activation, DNA fragmentation profile, chromatin morphology, and dependence on de novo gene expression. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, and dominant negatives and inhibitors of the p38 pathway prevent this apoptosis. In contrast, withdrawal of trophic support induces caspase-dependent death accompanied by JNK-dependent phosphorylation of c-Jun, and inhibition of JNK is sufficient to prevent the death induced by withdrawal of trophic support. Inhibition of p38 does not block withdrawal of trophic support-induced death, nor does inhibition of JNK block glutamate-induced death. We propose that mechanistically different forms of apoptosis have differing requirements for p38 and JNK activities in neurons and demonstrate that only inhibition of the appropriate kinase will prevent neurons from undergoing apoptosis.

Lithium has been used as an effective mood-stabilizing drug for the treatment of manic episodes and depression for 50 years. More recently, lithium has been found to protect neurons from death induced by a wide array of neurotoxic insults. However, the molecular basis for the prophylactic effects of lithium have remained obscure. A target of lithium, glycogen synthase kinase 3 (GSK-3), is implicated in neuronal death after trophic deprivation. The mechanism whereby GSK-3 exerts its neurotoxic effects is also unknown. Here we show that lithium blocks the canonical c-Jun apoptotic pathway in cerebellar granule neurons deprived of trophic support. This effect is mimicked by the structurally independent inhibitors of GSK-3, FRAT1, and indirubin. Like lithium, these prevent the stress induced c-Jun protein increase and subsequent apoptosis. These events are downstream of c-Jun transactivation, since GSK-3 inhibitors block neuronal death induced by constitutively active c-Jun (Ser/Thr-->Asp) and FRAT1 expression inhibits AP1 reporter activity. Consistent with this, AP1-dependent expression of proapoptotic Bim requires GSK-3-like activity. These data suggest that a GSK-3-like kinase acts in tandem with c-Jun N-terminal kinase to coordinate the full execution of the c-Jun stress response and neuronal death in response to trophic deprivation.

c-Jun is considered a major regulator of both neuronal death and regeneration. Stress in primary cultured CNS neurons induces phosphorylation of c-Jun serines 63 and 73 and increased c-Jun protein. However, total c-Jun N-terminal protein kinase (JNK) activity does not increase, and no satisfactory explanation for this paradox has been available. Here we demonstrate that neuronal stress induces strong activation of JNK2/3 in the presence of constitutively and highly active JNK1. Correspondingly, neurons from JNK1(-/-) mice show lower constitutive activity and considerably higher responsiveness to stress. p38 activity can be completely inhibited without effect on c-Jun phosphorylation, whereas 10 micrometer SB203580 strongly inhibits neuronal JNK2/3, stress-induced c-Jun phosphorylation, induced c-Jun activity, and neuronal death in response to trophic withdrawal stress. Neither constitutive JNK1 activity nor total neuronal JNK activity were significantly affected by this concentration of drug. Thus, neuronal stress selectively activates JNK2/3 in the presence of mechanisms maintaining constitutive JNK1 activity, and this JNK2/3 activity selectively targets c-Jun, which is isolated from constitutive JNK1 activity.

NMDA receptors (NMDAR) are glutamate-gated calcium channels that play pivotal roles in fundamental aspects of neuronal function. Dysregulated receptor function contributes to many disorders. Recruitment by NMDARs of calcium-dependent enzyme nNOS via PSD95 is seen as a key contributor to neuronal dysfunction. nNOS adaptor protein (NOS1AP), originally described as a competitor of PSD95:nNOS interaction, is regarded an inhibitor of NMDAR-driven nNOS function. In conditions of NMDAR hyperactivity such as excitotoxicity, one expects NOS1AP to be neuroprotective. Conditions of NMDAR hypoactivity, as thought to occur in schizophrenia, might be exacerbated by NOS1AP. Indeed GWAS have implicated NOS1AP and nNOS in schizophrenia. Several studies now indicate NOS1AP can mediate rather than inhibit NMDAR/nNOS-dependent responses, including excitotoxic signaling. Yet the concept of NOS1AP as an inhibitor of nNOS predominates in studies of human disease genetics. Here we review the experimental evidence to evaluate this apparent controversy, consider whether the known functions of NOS1AP might defend neurons against NMDAR dysregulation and highlight specific areas for future investigation to shed light on the functions of this adaptor protein. [ABSTRACT FROM AUTHOR]

Normal functioning of the nervous system requires precise regulation of dendritic shape and synaptic connectivity. Here, we report a severe impairment of dendritic structures in the cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an unbiased screen for candidate mediators, we identify the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2 proline-rich domain phosphorylation is decreased in JNK1-/- brain. We developed compartment-targeted JNK inhibitors to define whether a functional relationship exists between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process elongation is enhanced after activation of JNK. Using JNK1-/- neurons, we reveal a dominant role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates dendrite shape under conditions in which JNK activity is low (JNK1-/- neurons). These results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of MAP2 plays an important role in defining dendritic architecture in the brain. [ABSTRACT FROM AUTHOR]

Abstract Neurotoxicity is one of the side-effects of the therapeutically useful antitumour agent, Ara-C (or 1-beta-d-arabinofuranosyl-cytosine, cytarabine). This agent is also reported to induce cell death of cultured neurons. In this study, we show that Ara-C-induced death of differentiating rat cerebellar granule neurons is prevented by cycloheximide at concentrations corresponding to its action in preventing protein synthesis. The death is accompanied by cleavage of the caspase substrate poly ADP ribose polymerase (PARP) and c-Abl-dependent activation of the stress-activated protein kinases c-Jun N-terminal kinase and p38. However, c-Jun levels do not rise and the activation of the stress-activated protein kinases is not required for this form of neuronal death. Cyclin-dependent kinase (cdk) activity and inappropriate cell-cycle re-entry have been implicated in some forms of death in differentiated neurons. Here we show that Ara-C-induced death of cerebellar granule neurons is prevented by an inhibitor of cdk4, whereas inhibition of cdk1, -2 and -5 mimics the death, and non-cdk4/6 cdks are inhibited by Ara-C treatment. Cdk1 and -2 are dramatically down-regulated during neuronal differentiation, and neither Ara-C nor inhibition of these cdks induces death in mature neurons. This mechanism could also play a significant role in the neurotoxicity associated with the therapeutic use of Ara-C, as cdk levels can be upregulated in stressed neurons of adult brain. We propose that the balance between cdk4/6 and cdk1/2/5 activity may determine the survival of early differentiating neurons, and that DNA-damaging agents may induce neuronal death by inhibiting cdk1/2/5 under conditions which require these activities for survival. [ABSTRACT FROM AUTHOR]