Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening

<p>Abstract</p> <p>Background</p> <p>Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems.</p> <p>Methods</p> <p>HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.</p> <p>Results</p> <p>Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line.</p> <p>Conclusion</p> <p>A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.</p

Lingual deficits in neurotrophin double knockout mice

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF −/− and wild-type mice. Taste papillae morphology was severely distorted in BDNF −/− x NT-3 −/− mice compared to single BDNF −/− and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF −/− and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF −/− x NT-3 −/− mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF −/− x NT-3 −/− mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47465/1/11068_2005_Article_3330.pd

Specificity of neurotrophins in the nervous system : A genetic approach to determine receptor engagement by neurotrophins

The classical neurotrophic factor hypothesis describes the neurotrophins as retrograde signalling factors supporting the survival of postmitotic neurons during the development. Knock-out mice for the neurotrophins and/or their receptors have been generated and their analyses gave new insights in the temporal expression and particular functions of these factors during development. Despite partially overlapping expression of neurotrophins and Trk receptor in both the central and peripheral nervous system, the different knock-outs display distinct phenotypes, demonstrating diverse and specific roles of the neurotrophins. Further analysis showed that several neurotrophins can function as survival factors for the same neurons, indicating a potential for the neurotrophins to display compensatory effects. Further comparison of the loss of neuronal numbers between neurotrophin and Trk receptor knock-outs have suggested a role for NT3 signalling through its non-preferred receptors, TrkA and TrkB. In this thesis the specificity and selective roles of BDNF and NT3 in the peripheral and central nervous system have been analysed. In order to analyse this, the coding part of the BDNF gene has been replaced by NT3, in genetically modified mice (BDNNT3/NT3). Analysis of the nervous system revealed striking differences in the ability of NT3 to replace BDNF. We conclude that there is no general mechanism by which neurotrophin specificity is attained and the specificity of neurotrophin signalling is set by several factors, which are dependent on the cellular system studied. These factors include a spatial and temporal expression of ligands and receptors and the activation of diverse intracellular pathways by different Trk receptors within the same neuronal population. Finally, we also investigated the ability of NT3 to signal through non-preferred receptors in vivo. We see that there is a high degree of receptor specificity between neurotrophins and their cognate receptors in vivo

Genetic evidence for selective neurotrophin 3 signalling through TrkC but not TrkB in vivo

Neurotrophins control neuronal survival in a target-derived manner during the period of naturally occurring cell death in development. The specificity of this mechanism has been attributed to a restricted spatio-temporal expression of neurotrophin ligands in target tissues, as well as a selective expression of their cognate tyrosine kinase (Trk) receptors in different neuronal subpopulations. However, several in vitro and in vivo studies of null mutant mice have suggested that neurotrophin 3 (NT3) also signals through the non-preferred TrkB receptor. In this study, we have directly addressed the in vivo preference of NT3 to signal through TrkB or TrkC, by crossing the NT3 knock-in mice (BDNF(NT3/NT3) mice) with the TrkB- or TrkC-null mutant mice. We find that TrkB is dispensable, whereas TrkC is required for the neuronal rescue by the NT3 allele in the brain-derived neurotrophic factor- and NT3-dependent cochleovestibular system. Our results show that NT3 maintains survival of cells as well as target innervation only through interactions with TrkC in vivo. TrkB and TrkC receptors are thus not functionally redundant for NT3, even when coexpressed in neurons of the cochleovestibular system

Gene Therapy Vector Encoding Neuropeptide Y and Its Receptor Y2 for Future Treatment of Epilepsy:Preclinical Data in Rats

Gene therapy to treat pharmacoresistant temporal lobe epilepsy in humans is now being developed using an AAV vector (CG01) that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2. With this in mind, the present study aimed to provide important preclinical data on the effects of CG01 on the duration of transgene expression, cellular tropism, and potential side effects on body weight and cognitive function. The CG01 vector was administered unilaterally into the dorsal and ventral hippocampus of adult male rats and expression of both transgenes was found to remain elevated without a sign of decline at 6 months post-injection. CG01 appeared to mediate expression selectively in hippocampal neurons, without expression in astrocytes or oligodendrocytes. No effects were seen on body weight as well as on short- or long-term memory as revealed by testing in the Y-maze or Morris water maze tests. Thus these data show that unilateral CG01 vector treatment as future gene therapy in pharmacoresistant temporal lobe epilepsy patients should result in stable and long-term expression predominantly in neurons and be well tolerated without side effects on body weight and cognitive function