Biomedical subjects

Ashok B Kulkarni

Publications and source records attributed to Ashok B Kulkarni.

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

Neuron-specific TGF-beta signaling deficiency results in retinal detachment and cataracts in mice.

We generated a mouse model (cKO) with a conditional deletion of TGF-beta signaling in the retinal neurons by crossing TGF-beta receptor I (TGF-beta RI) floxed mice with nestin-Cre mice. Almost all of the newborn cKO mice had retinal detachment at the retinal pigment epithelium (RPE)/photoreceptor layer junction of the neurosensory retina (NSR). The immunostaining for chondroitin-6-sulfate showed a very weak reaction in cKO mice in contrast to intense staining in the photoreceptor layer in wild-type mice. Macroscopic cataracts, in one or both eyes, were observed in 50% of the mice by 6 months of age, starting as early as the first month after birth. The cKO mouse model demonstrates that the TGF-beta signaling deficiency in retinal cells leads to decreased levels of chondroitin sulfate proteoglycan in the retinal interphotoreceptor matrix. This in turn causes retinal detachment due to the loss of adhesion of the NSR to RPE.

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

Cdk5: a new player in pain signaling.

Cyclin-dependent kinase 5 (Cdk5) is predominantly active in postmitotic neurons. Despite its structural homology with other cyclin-dependent kinases, Cdk5 is apparently not involved in the cell cycle process. The monomeric form of Cdk5 is inactive and requires the association of p35 or p39 in order to perform its kinase activity. This kinase is essential for normal brain development and function, but uncontrolled activity of Cdk5 may lead to numerous neurodegenerative processes. Although Cdk5 activity has been implicated in several neuronal functions, its precise role in the peripheral nervous system has not been determined. Recently we reported for the first time the essential role for Cdk5 in pain signaling (Pareek et al., PNAS 2006; 103:791-6). Altered nociceptive responses to basal thermal noxious stimuli in p35 knockout (p35(-/-)) and p35-overexpresing transgenic mice (Tgp35) have established the important role of this gene in the nociceptive process. Here, we report that Cdk5 regulates mitogen-activated protein kinase kinase1/2 (MEK1/2) activity through a negative feedback loop during the peripheral inflammatory response. Moreover a differential nociceptive response after chronic morphine exposure in p35(-/-) and Tgp35 mice suggests that Cdk5 activity is important for opioid tolerance. In conclusion, our data indicate important molecular roles for Cdk5 in pain signaling and opioid tolerance, which makes it a potential target for analgesic drug development.

Analgesics, Opioid↗

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

Overexpression of transforming growth factor-beta1 in teeth results in detachment of ameloblasts and enamel defects.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

Ameloblasts↗

PubMed2006

Comparison of body weight and gene expression in amelogenin null and wild-type mice.

Amelogenin (AmelX) null mice develop hypomineralized enamel lacking normal prism structure, but are healthy and fertile. Because these mice are smaller than wild-type mice prior to weaning, we undertook a detailed analysis of the weight of mice and analyzed AmelX expression in non-dental tissues. Wild-type mice had a greater average weight each day within the 3-wk period. Using reverse transcription-polymerase chain reaction (RT-PCR), products of approximately 200 bp in size were generated from wild-type teeth, brain, eye, and calvariae. DNA sequence analysis of RT-PCR products from calvariae indicated that the small amelogenin leucine-rich amelogenin peptide (LRAP), both with and without exon 4, was expressed. No products were obtained from any of the samples from the AmelX null mice. We also isolated mRNAs that included AmelX exons 8 and 9, and identified a duplication within the murine AmelX gene with 91% homology. Our results add additional support to the hypothesis that amelogenins are multifunctional proteins, with potential roles in non-ameloblasts and in non-mineralizing tissues during development. The smaller size of AmelX null mice could potentially be explained by the lack of LRAP expression in some of these tissues, leading to a delay in development.

Amelogenin↗

PubMed2006

A developmental comparison of matrix metalloproteinase-20 and amelogenin null mouse enamel.

Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.

Absorption↗

PubMed2006

Dph3, a small protein required for diphthamide biosynthesis, is essential in mouse development.

The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3+/- mice are phenotypically normal, whereas dph3-/- mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3-/- placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.

Adenosine Diphosphate↗

PubMed2005

The KIF3 motor transports N-cadherin and organizes the developing neuroepithelium.

In the developing brain, the organization of the neuroepithelium is maintained by a critical balance between proliferation and cell-cell adhesion of neural progenitor cells. The molecular mechanisms that underlie this are still largely unknown. Here, through analysis of a conditional knockout mouse for the Kap3 gene, we show that post-Golgi transport of N-cadherin by the KIF3 molecular motor complex is crucial for maintaining this balance. N-cadherin and beta-catenin associate with the KIF3 complex by co-immunoprecipitation, and colocalize with KIF3 in cells. Furthermore, in KAP3-deficient cells, the subcellular localization of N-cadherin was disrupted. Taken together, these results suggest a potential tumour-suppressing activity for this molecular motor.

Adaptor Proteins, Signal Transducing↗

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

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↗

PubMed2005

The use of animal models to explore amelogenin variants in amelogenesis imperfecta.

Amelogenin proteins are secreted by ameloblast cells during tooth development. Because of extensive alternative splicing of the amelogenin primary RNA transcript, and because systematic proteolysis results in many additional peptides during enamel maturation, it has been difficult to assign function to individual amelogenins. Targeted deletions and transgenic mice have been generated in order to better understand amelogenin protein function in vivo. From these murine models, we have determined that amelogenins are responsible for normal enamel thickness and structure, but not for initiation of enamel mineral formation at the dentin-enamel junction. Although it is now clear that the amelogenin (AmelX) gene exists in a nested orientation and that AmelX is expressed at a low level in various developing tissues, the significance of these findings is incompletely understood. Future studies are expected to answer remaining questions concerning structure/function relationships among these 'enamel proteins'.

Amelogenesis Imperfecta↗

PubMed2004

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Ameloblasts↗

PubMed2004

Transgenic mouse expressing human mutant alpha-galactosidase A in an endogenous enzyme deficient background: a biochemical animal model for studying active-site specific chaperone therapy for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by the deficiency of lysosomal alpha-galactosidase A (alpha-Gal A). We have established transgenic mice that exclusively express human mutant alpha-Gal A (R301Q) in an alpha-Gal A knock-out background (TgM/KO mice). This serves as a biochemical model to study and evaluate active-site specific chaperone (ASSC) therapy for Fabry disease, which is specific for those missense mutations that cause misfolding of alpha-Gal A. The alpha-Gal A activities in the heart, kidney, spleen, and liver of homozygous TgM/KO mice were 52.6, 9.9, 29.6 and 44.4 unit/mg protein, respectively, corresponding to 16.4-, 0.8-, 0.6- and 1.4-fold of the endogenous enzyme activities in the same tissues of non-transgenic mice with a similar genetic background. Oral administration of 1-deoxygalactonojirimycin (DGJ), a competitive inhibitor of alpha-Gal A and an effective ASSC for Fabry disease, at 0.05 mM in the drinking water of the mice for 2 weeks resulted in 13.8-, 3.3-, 3.9-, and 2.6-fold increases in enzyme activities in the heart, kidney, spleen and liver, respectively. No accumulation of globotriaosylceramide, a natural substrate of alpha-Gal A, could be detected in the heart of TgM/KO mice after DGJ treatment, indicating that degradation of the glycolipid in the heart was not inhibited by DGJ at that dosage. The alpha-Gal A activity in homozygous or heterozygous fibroblasts established from TgM/KO mice (TMK cells) was approximately 39 and 20 unit/mg protein, respectively. These TgM/KO mice and TMK cells are useful tools for studying the mechanism of ASSC therapy, and for screening ASSCs for Fabry disease.

1-Deoxynojirimycin↗

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↗

PubMed2004

Cdk5 phosphorylation of doublecortin ser297 regulates its effect on neuronal migration.

Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.

Animals↗

PubMed2004

Cyclin-dependent kinase 5 is a regulator of podocyte differentiation, proliferation, and morphology.

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cyclin-dependent kinase 5 (CDK5) has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. In this study, we examined the role of CDK5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, CDK5 increased in association with podocyte differentiation. During mouse glomerulogenesis in vivo, CDK5 expression was predominantly detected in podocytes from the capillary loop stage to maturation and persisted in the podocytes of adult glomeruli. In contrast, CDK5 was markedly decreased in the proliferating and dedifferentiated podocytes of mice with anti-glomerular basement membrane nephritis and in human immunodeficiency virus transgenic mice. p35, the activator of CDK5, was also detected in podocytes and the p35/CDK5 complex was active. Cell fractionation studies showed that active p35/CDK5 was mainly localized to the plasma membrane. Specific inhibition of CDK5 in differentiated cultured podocytes, either pharmacologically or with siRNA, induced shape changes, with cellular elongation and loss of process formation compared to the characteristic arborized phenotype. These data suggest a role for CDK5 as a regulator of podocyte differentiation, proliferation, and morphology.

Animals↗