Biomedical subjects

Harish C Pant

Publications and source records attributed to Harish C Pant.

At least 19 recordsLinked to original sources

PubMed2006

Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.

Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. The phosphorylation of TRPV1 provides a versatile regulation of intracellular calcium levels and is critical for TRPV1 function in responding to a pain stimulus. We have previously reported that cyclin-dependent kinase 5 (Cdk5) activity regulates nociceptive signaling. In this article we report that the Cdk5-mediated phosphorylation of TRPV1 at threonine-407 can modulate agonist-induced calcium influx. Inhibition of Cdk5 activity in cultured dorsal root ganglia neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional-knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. Thus, the present study indicates that Cdk5-mediated TRPV1 phosphorylation is important in the regulation of pain signaling.

Amino Acid Sequence↗

PubMed2006

Cloning and characterization of zebrafish (Danio rerio) cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by neuron-specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in primary neurogenesis remains unexplored. Here, we have cloned and characterized the zebrafish cdk5 ortholog. Zebrafish cdk5 is 96% identical to its human counterpart. In situ hybridization analyses demonstrated that zebrafish cdk5 transcripts are ubiquitously expressed as early as the blastula stage. At 11.5h of development, cdk5 transcripts were present in the neural plate at the domains where primary neurons begin to be specified. RT-PCR analyses showed equal levels of cdk5 transcripts up to 72 h of development. SiRNA-mediated cdk5 knockdown resulted in a reduction in primary sensory neurons of the trigeminal ganglia of the peripheral nervous system, suggesting that cdk5 plays a crucial role in the development of the peripheral nervous system.

Animals↗

PubMed2006

Cdk5 Modulation of mitogen-activated protein kinase signaling regulates neuronal survival.

Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.

Animals↗

PubMed2006

Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide releasing factor-1.

RasGRF1 is a member of the Ras guanine nucleotide exchange factor (RasGEF) family of proteins which are directly responsible for the activation of Ras and Rac GTPases. Originally identified as a phosphoprotein, RasGRF1 has been shown to be phosphorylated by protein kinase A and more recently, by the non-receptor tyrosine kinases Ack1 and Src. In this report we show that RasGRF1 interacts with and is phosphorylated by Cdk5 on serine 731 to regulate its steady state levels in mammalian cells as well as in neurons. Phosphorylation on this site by Cdk5 leads to RasGRF1 degradation through a calpain-dependent mechanism. Additionally, cortical neurons from Cdk5 knockout mice have higher levels of RasGRF1 which are reduced when wild-type Cdk5 is transfected into these neurons. In mitotic cells, nuclei become disorganized when RasGRF1 is overexpressed and this is rescued when RasGRF1 is co-expressed with active Cdk5. When RasGRF1 levels are elevated in neurons through overexpression of either the wild-type RasGRF1, or the phosphorylation mutant of RasGRF1 and by the transfection of a dominant negative Cdk5 construct, nuclei appeared condensed and fragmented. On the other hand, a reduction of RasGRF1 levels through p35/Cdk5 overexpression also leads to nuclear condensation in neurons. These data show that phosphorylation of RasGRF1 by Cdk5 tightly regulates its levels, which is essential for proper cellular organization.

Animals↗

PubMed2006

Cyclin-dependent kinase 5 influences Rohon-Beard neuron survival in zebrafish.

Cyclin-dependent kinase 5 (cdk5), a member of the cyclin-dependent kinase family, is expressed predominantly in post-mitotic cell populations. Unlike the other cdks, cdk5 is abundant and most active in differentiated neurons. Here, we describe the function of a cdk5 ortholog in zebrafish. Cdk5 catalytic activity is meager but present in early stages of development. However, at 24 h post-fertilization (hpf), the activity is remarkably higher and continues to be high through 48 and 72 hpf. Knocking down cdk5 by micro-injection of a specific siRNA resulted in decreased cdk5 protein level accompanied by reduced kinase activity. In the cdk5 siRNA-injected embryos, the number of primary sensory Rohon-Beard (RB) neurons was significantly reduced and there were more apoptotic cells in the brain. These phenotypes were rescued by co-injection of cdk5 mRNA. Within the first two days of development, RB neurons undergo apoptosis in zebrafish. To examine whether cdk5 has a role in RB neuron survival, cdk5 mRNA was injected into the one- to two-cell embryos. In these embryos, RB neuron apoptosis was inhibited compared with the uninjected control embryos. These results suggest that in zebrafish, cdk5 influences RB neuron survival and potentially regulates early neuronal development.

Animals↗

PubMed2006

Cyclin-dependent kinase 5 activity regulates pain signaling.

Several molecules and cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in the development and disease of the mammalian nervous system. The precise role of this kinase in sensory pathways has not been well characterized. Here we report a molecular role for Cdk5 in nociception. We identified the expression of Cdk5 and its activator p35 in nociceptive neurons, which is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons after inflammation resulted in the cleavage of p35 to p25, which forms a more stable complex with Cdk5 and, consequently, leads to elevation of Cdk5 activity. p35 knockout mice (p35(-/-)), which exhibit significantly decreased Cdk5 activity, showed delayed responses to painful thermal stimulation compared with WT controls. In contrast, mice overexpressing p35, which exhibit elevated levels of Cdk5 activity, were more sensitive to painful thermal stimuli than were controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for the development of analgesic drugs.

Animals↗

PubMed2006

Squid (Loligo pealei) giant fiber system: a model for studying neurodegeneration and dementia?

In many neurodegenerative disorders that lead to memory loss and dementia, the brain pathology responsible for neuronal loss is marked by accumulations of proteins in the form of extracellular plaques and intracellular filamentous tangles, containing hyperphosphorylated cytoskeletal proteins. These are assumed to arise as a consequence of deregulation of a normal pattern of topographic phosphorylation-that is, an abnormal shift of cytoskeletal protein phosphorylation from the normal axonal compartment to cell bodies. Although decades of studies have been directed to this problem, biochemical approaches in mammalian systems are limited: neurons are too small to permit separation of cell body and axon compartments. Since the pioneering studies of Hodgkin and Huxley on the giant fiber system of the squid, however, the stellate ganglion and its giant axons have been the focus of a large literature on the physiology and biochemistry of neuron function. This review concentrates on a host of studies in our laboratory and others on the factors regulating compartment-specific patterns of cytoskeletal protein phosphorylation (primarily neurofilaments) in an effort to establish a normal baseline of information for further studies on neurodegeneration. On the basis of these data, a model of topographic regulation is proposed that offers several possibilities for further studies on potential sites of deregulation that may lead to pathologies resembling those seen in mammalian and human brains showing neurodegeneration, dementia, and neuronal cell death.

Animals↗

PubMed2005

Cdk5 inhibits anterograde axonal transport of neurofilaments but not that of tau by inhibition of mitogen-activated protein kinase activity.

Cyclin-dependent kinase 5 (cdk5) inhibits neurofilament (NF) anterograde axonal transport while p42/44 mitogen-activated protein kinase (MAPk) promotes it. Since cdk5 is known to inhibit MAP kinase activity, we examined whether or not cdk5 inhibits anterograde NF transport via inhibition of MAPk activity. To accomplish this, we manipulated the activity of these kinases in differentiated NB2a/d1 cells, and monitored anterograde axonal transport of green fluorescent protein-conjugated-NF-M (GFP-M) and cyan fluorescent protein-conjugated (CFP)-tau. The cdk5 inhibitor roscovitine increased anterograde axonal transport of GFP-M and CFP-tau; transfection with cdk5/p25 inhibited transport of both. Inhibition of MAPk activity by PD98059 or expression of dominant-negative MAPk inhibited anterograde GFP-M transport, while expression of constitutively active MAPk enhanced it; these treatments did not affect CFP-tau transport. PD98059 prevented roscovitine-mediated enhancement of GFP-M transport, but did not prevent enhancement of CFP-tau transport. Co-transfection with constitutively activated MAPk prevented the inhibition of GFP-M transport that normally accompanied transfection with cdk5/p25, but did not prevent inhibition of tau transport by cdk5/p25. Finally, the extent of inhibition of GFP-M axonal transport by PD98059 was not additive to that derived from transfection with cdk5/p35, and the increase in NF transport that accompanies roscovitine treatment was not additive to that derived from transfection with constitutively activated MAPk, suggesting that the influence of these kinases on NF transport was within the same, rather than distinct, pathways. These findings suggest that axonal transport of tau and NFs is under the control of distinct kinase cascades, and that cdk5 inhibits NF transport at least in part by inhibiting MAPk.

Animals↗

PubMed2005

Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling.

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.

Animals↗

PubMed2005

Nuclear localization of Munc18-1 (p67) in the adult rat brain and PC12 cells.

Munc18-1, also referred to as p67, co-purifies with Cdk5 and has an important role in neurotransmitter release. The role of Munc18-1 for functional connectivity of the nervous system was demonstrated by gene knockout experiments in mice, wherein accumulation of neurotransmitter and silencing of synaptic activity was observed. Our earlier studies have shown that both Munc18-1 and Cdk5 co-purify and co-localize with cytoskeletal components, implying that apart from having a regulatory role in vesicle docking and fusion, Munc18-1 could also affect the dynamics of neuronal cytoskeleton. In the present study we have shown the presence of Munc18-1 in nuclear rich fraction from rat brain and confirmed the nuclear localization of this protein in PC12 cells and adult rat brain neurons by immunofluorescence and immunoelectron microscopy. We also demonstrate the binding of Munc18-1 to double stranded (ds) DNA. The ability of Munc18-1 to bind dsDNA, albeit the lack of DNA binding domains, suggests that the binding may be mediated through protein-protein interaction through some other DNA-binding proteins. The presence of both nuclear import and export signals in Munc18-1 primary structure corroborates its nuclear localization and makes it a putative shuttle protein between nuclear and cytoplasmic compartments, the precise physiological relevance of which needs to be elucidated.

Active Transport, Cell Nucleus↗

PubMed2005

The pathobiology of amyotrophic lateral sclerosis: a proteinopathy?

Amyotrophic lateral sclerosis (ALS) is increasingly considered to be a disorder of multiple etiologies that have in common progressive degeneration of both upper and lower motor neurons, ultimately giving rise to a relentless loss of muscle function. This progressive degeneration is associated with heightened levels of oxidative injury, excitotoxicity, and mitochondrial dysfunction--all occurring concurrently. In this article, we review the evidence that suggests, in common with other age-dependent neurodegenerative disorders, that ALS can be considered a disorder of protein aggregation. Morphologically, this is evident as Bunina bodies, ubiquitin-immunoreactive fibrils or aggregates, neurofilamentous aggregates, mutant copper/zinc superoxide dismutase (SOD1) aggregates in familial ALS variants harboring mutations in SOD1, peripherin-immunoreactive aggregates within spinal motor neurons and as neuroaxonal spheroids, and in an increasingly greater population of patients with ALS with cognitive impairment, both intra- and extraneuronal tau aggregates. We review the evidence that somatotopically specific patterns of altered kinase and phosphatase activity are associated with alterations in the phosphorylation state of these proteins, altering either solubility or assembly characteristics. The role of nonneuronal cells in mediating motor neuronal injury is discussed in the context of alterations in tyrosine kinase activity and enhanced protein phosphorylation.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability.

Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca(2+)/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

Animals↗

PubMed2004

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons.

The extracellular aggregation of amyloid beta (Abeta) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Abeta peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Abeta(1-42)-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

Amyloid beta-Peptides↗

PubMed2004

Mitogen-activated protein kinase regulates neurofilament axonal transport.

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.

Animals↗

PubMed2004

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress.

Cyclin-dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho-NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho-NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5-mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein-tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.

Animals↗

PubMed2004

A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons.

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane-bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule-dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin-driven motility in axons. This pathway involves enzymatic activities of cyclin-dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.

Animals↗

PubMed2004

p35/cyclin-dependent kinase 5 phosphorylation of ras guanine nucleotide releasing factor 2 (RasGRF2) mediates Rac-dependent Extracellular Signal-regulated kinase 1/2 activity, altering RasGRF2 and microtubule-associated protein 1b distribution in neurons.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in "cross-talk" with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac-guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.

Animals↗

PubMed2004

Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects.

Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.

Animals↗