Locomotor activity and evoked dopamine release are reduced in mice overexpressing A30P-mutated human alpha-synuclein.

We have generated a transgenic mouse line overexpressing mutated human A30P alpha-synuclein under the control of the prion-related protein promoter. Immunohistology revealed mutated human A30P alpha-synuclein protein in numerous brain areas, but no gross morphological changes, Lewy bodies, or loss of dopaminergic cell bodies. The transgenic mice displayed decreased locomotion, impaired motor coordination, and balance. In vivo voltammetry showed that A30P mice responded to longer stimulation of the ascending dopaminergic pathways with less dopamine release in striatum and had a slower rate of dopamine decline after repeated stimulations or after alpha-methyl-p-tyrosine-HCl treatment. However, dopamine re-uptake or transporter levels were similar in transgenic and control mice. Our data provide evidence that overexpression of mutated human A30P alpha-synuclein in mice leads to a reduced size of the dopamine storage pool. This is in agreement with the previously postulated involvement of alpha-synuclein in the turnover of transmitter vesicles and may explain the observed motor deficits in A30P mice.

Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediate glutamate-induced cleavage of the cyclin-dependent kinase 5 (cdk5) activator p35 in cultured rat hippocampal neurons.

Cyclin-dependent kinase 5 (cdk5) regulates crucial neurobiological events, and deregulation of cdk5 has been implicated in several neurodegenerative disorders. The deregulation is suggested to occur due to cleavage of the cdk5 activator protein p35 to a smaller p25 fragment by the calcium-activated protease calpain. Here we have elucidated the role of different calcium-permeable ionotropic glutamate receptors in the induction of p35 cleavage in cultured rat hippocampal neurons. The glutamate receptor agonists glutamic acid, N-methyl-D-aspartate (NMDA), kainic acid, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were all able to induce p35 cleavage, in a manner depending on extracellular calcium. The effect of glutamate was mediated by NMDA receptors, as it was prevented by the NMDA antagonist MK-801. Cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization, enhanced glutamate-induced p35 cleavage. In immature 6-day-old cultures the non-NMDA agonist kainic acid provoked p35 cleavage, whereas glutamate and NMDA were ineffective. The data suggest that both NMDA and non-NMDA receptors are able to induce p35 cleavage. Different factors, such as maturation state of neurons or desensitization properties of non-NMDA receptors, may determine which receptor predominantly mediates the effect of glutamate on p35 cleavage.

Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation.

Hyperphosphorylated tau protein is the primary component of neurofibrillary tangles observed in several neurodegenerative disorders. It has been hypothesized that in certain pathological conditions, the calcium activated protease, calpain, would cleave the cyclin-dependent kinase 5 (cdk5) activator p35 to a p25 fragment, which would lead to augmented cdk5 activity, and cdk5-mediated tau hyperphosphorylation. To test this hypothesis, we induced calpain-mediated p35 cleavage in rat hippocampal neuronal cultures and studied the relationship between p25 production, cdk5 activity, and tau phosphorylation. In glutamate-treated cells p35 was cleaved to p25 and this was associated with elevated cdk5 activity. However, tau phosphorylation was concomitantly decreased at multiple sites. The calpain inhibitor MDL28170 prevented the cleavage of p35 but had no effect on tau phosphorylation, suggesting that calpain-mediated processes, i.e., the cleavage of p35 to p25 and cdk5 activation, do not contribute to tau phosphorylation in these conditions. Treatment of the neuronal cultures with N-methyl-D-aspartic acid or with calcium ionophores resulted in an outcome highly similar to that of glutamate. We conclude that, in neuronal cells, the cleavage of p35 to p25 is associated with increased activity of cdk5 but not with tau hyperphosphorylation.

Influence of phosphorylation of p35, an activator of cyclin-dependent kinase 5 (cdk5), on the proteolysis of p35.

Cyclin-dependent kinase 5 (cdk5) is involved in the development of the nervous system and neuronal process outgrowth, and it regulates several intracellular processes including cytoskeletal dynamics. Dysregulation of cdk5 has been implicated in many disorders of the nervous system. The activity of the kinase is regulated by binding of cdk5 activators (p35, p39, p67). We examined the phosphorylation of p35, and the role of phosphorylation in regulating the proteolysis of the p35 protein. By detecting changes in electrophoretic mobility, we observed that a significant proportion of p35 is phosphorylated in rat brain tissue. In cultured neurons, the phosphorylation was prevented by roscovitine, an inhibitor of cdk5 and some other cdks. The phosphatase inhibitor okadaic acid induced p35 degradation in neuronal cultures which was sensitive to the proteasome inhibitor lactacystin. These latter results agree with some previous studies showing that phosphorylation regulates proteasomal degradation of p35. Treatment of brain homogenate with okadaic acid in the presence of ATP led to accumulation of p35 phosphorylated also by a kinase that was not inhibited by roscovitine. This implies that the effect of okadaic acid on p35 degradation could also be contributed by a non-cdk kinase. The calpain protease has been shown to cleave p35. Our results suggest that this process may also be modulated by p35 phosphorylation under some conditions. We conclude that p35 phosphorylation influences the proteasome-mediated degradation of p35 and calpain-mediated cleavage of p35 to p25.