Jyotshnabala Kanungo

Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys245–Ala277. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.

Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the “four-eyed” fish (Anableps anableps)

The water meniscus bisects the eyes of the “four‐eyed” fish Anableps anableps, resulting in simultaneous vision in air and water. We compare the structure and macromolecular compositions of the Anableps dorsal (air) and ventral (water) corneas with the fully aquatic zebrafish cornea. The Anableps dorsal corneal epithelium is thicker (>20 cell layers), flatter (∼1.94 mm radius of curvature), and contains ∼15‐fold more glycogen (0.16 µg/µg water‐soluble protein) than the ventral corneal epithelium (5–7 cell layers; ∼1.63 mm radius of curvature; 0.01 µg glycogen/µg water‐soluble protein), which resembles the zebrafish corneal epithelium. Gelsolin is the major water‐soluble protein in the zebrafish (∼50%) and Anableps dorsal (∼38%) and ventral (∼21%) corneal epithelia, suggesting that gelsolin was recruited for high corneal expression before these two species diverged at least 100 million years ago and that abundant corneal gelsolin is not limited to aquatic vision. Anableps gelsolin, deduced from its cDNA, is 57% identical to zebrafish gelsolin. Paucity of Anableps corneal F‐actin (consistent with high gelsolin) was confirmed by the absence of rhodamine‐phalloidin staining. We suggest amphibious refraction and protection from UV irradiation and desiccation in air as selective constraints for the specializations of the Anableps dorsal cornea.—Swamynathan, S. K., Crawford, M. A., Robison, W. G. Jr., Kanungo, J., Piatigorsky, J. Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the “four‐eyed” fish (Anableps anableps). FASEB J. 17, 1996–2005 (2003)

Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration

The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca2+ activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.

Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin

We have previously identified a gelsolin‐like protein (C/L‐gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin‐like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L‐gelsolin) and scinlb are novel scinderin‐like genes. RT‐PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT‐PCR and 2‐dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of β‐actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3‐day‐old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L‐gelsolin.—Jia S., Omelchenko, M., Garland, D., Vasiliou, V., Kanungo, J., Spencer, M., Wolf, Y., Koonin, E., Piatigorsky J. Duplicated gelsolin family genes in zebrafish: a novel scinderin‐like gene (scinla) encodes the major corneal crystallin. FASEB J. 21, 3318–3328 (2007)

Phosphorylation of p27Kip1 at Thr187 by cyclin-dependent kinase 5 modulates neural stem cell differentiation.

Cdk5 plays a role in nervous system development; its role in the initial stages of neural differentiation is poorly understood. We isolated neural stem cells from E13 Cdk5 WT and KO mouse and observed them as they switched from proliferating stage to neural differentiation. We show that Cdk5 phosphorylation of p27kip1 at Thr187 is crucial to neural differentiation.

The Notch signaling inhibitor DAPT down-regulates cdk5 activity and modulates the distribution of neuronal cytoskeletal proteins.

Notch signaling is critical for the development of the nervous system. Cyclin‐dependent kinase 5 (cdk5) is a neuronal kinase involved in neuronal development and phosphorylates a number of neuronal cytoskeletal proteins. To determine the relationship between Notch and cdk5 signaling, we tested the effects of the Notch inhibitor, N‐[N‐(3,5‐difluorophenacetyl)‐1‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) on cdk5 expression, activity and cytoskeletal protein distribution in the rat cortical neurons in primary cultures. Neurons treated with 10 μM DAPT showed attenuated cdk5 activity in spite of an up‐regulation of cdk5 protein level, consistent with a phenomenon reported in the cdk5 transgenic mice. Immunoblot and immunofluorescence analyses showed an increased level of cdk5, but not p35. Phospho‐tau and phospho‐neurofilament showed a shift from axons to cell bodies in DAPT‐treated cells. DAPT‐induced attenuation of cdk5 activity was restored by over‐expression of p35 indicating that it interacted with cdk5 and up‐regulated nascent cdk5 activity. p35 over‐expression also rescued DAPT‐induced translocation of phospho‐tau and phospho‐neurofilament. Immunoprecipitation followed by immunoblotting demonstrated that DAPT does not disrupt cdk5 and p35 interaction. Moreover, DAPT up‐regulated neurogenin that is negatively regulated by Notch, and down‐regulated Hes1, a downstream target of Notch, suggesting that Notch signaling in the cortical neurons was disrupted. Semi‐quantitative and quantitative RT‐PCR analyses confirmed that DAPT up‐regulated cdk5 expression at the transcriptional level. These results establish a link between Notch signaling and cdk5 expression regulating neuronal cytoskeletal protein dynamics.

Gelsolin is a dorsalizing factor in zebrafish

The gene for gelsolin (an actin-binding, cytoskeletal regulatory protein) was shown earlier to be specialized for high corneal expression in adult zebrafish. We show here that zebrafish gelsolin is required for proper dorsalization during embryogenesis. Inhibition of gelsolin expression by injecting fertilized eggs with a specific morpholino oligonucleotide resulted in a range of concentration-dependent ventralized phenotypes, including those lacking a brain and eyes. These were rescued by coinjection of zebrafish gelsolin or chordin (a known dorsalizing agent) mRNAs, or human gelsolin protein. Moreover, injection of gelsolin mRNA or human gelsolin protein by itself dorsalized the developing embryos, often resulting in axis duplication. Injection of the gelsolin-specific morpholino oligonucleotide enhanced the expression of Vent mRNA, a ventral marker downstream of bone morphogenetic proteins, whereas injection of gelsolin mRNA enhanced the expression of chordin and goosecoid mRNAs, both dorsal markers. Our results indicate that gelsolin also modulates embryonic dorsal/ventral pattern formation in zebrafish.

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.

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.