Intermediate filament (IF) overproduction induces abnormal accumulation of neuronal IF, which is a pathological indicator of some neurodegenerative disorders. In our study, α-Internexin- and peripherin-overexpressing PC12 cells (pINT-EGFP and pEGFP-peripherin) were used as models to study neuropathological pathways responsible for neurodegenerative diseases. Microarray data revealed that Cdk5-related genes were downregulated and Cdk5 regulatory subunit-associated protein 3 (GSK-3α and GSK-3β) were upregulated in pINT-EGFP cells. Increased expression of phosphorylated neurofilament and aberrant activation of Cdk5 and GSK-3β were detected in both pEGFP-peripherin and pINT-EGFP cells by Western blotting. In addition, pharmacological approaches to retaining viability of pINT-EGFP and pEGFP-peripherin cells were examined. Treatment with Cdk5 inhibitor and GSK-3β inhibitor significantly suppressed neuronal death. Dynamic changes of disaggregation of EGFP-peripherin and decrease in green fluorescence intensity were observed in pEGFP-peripherin and pINT-EGFP cells by confocal microscopy after GSK-3β inhibitor treatment. We conclude that inhibition of Cdk5 and GSK-3β suppresses neurofilament phosphorylation, slows down the accumulation of neuronal IF in the cytoplasm, and subsequently avoids damages to cell organelles. The results suggest that suppression of extensive neurofilament phosphorylation may be a potential strategy for ameliorating neuron death. The suppression of hyperphosphorylation of neuronal cytoskeletons with kinase inhibitors could be one of potential therapeutic treatments for neurodegenerative diseases.

Cerebellar mechanisms were explored underlying the effects of amphetamine (Amph) on the brain, by monitoring primarily the neurochemistry of the cerebellum. Adult mice received repeated intermittent injections of d-Amph, 5mg/kg or saline, twice daily for three days and once on day 4. As revealed by the biochemical analysis, the levels of GABA content were increased by 68-93% in the cerebellar vermis and hemisphere of mice at 4h after the first (acute) or the last (repeated) Amph injections, though the glutamate content was unaltered, compared to the respective saline-treated controls. By contrast, at 4h post-repeated Amph, in the vermis, the level of norepinephrine was approximate 38% lower than the corresponding control and 5-hydroxytryptamine (5-HT) resembled the control, whereas in the hemisphere, the norepinephrine content was similar to control and 5-HT 66% higher, implying cerebellar lobe-dependent changes. However, in both lobes, at 4h after the acute and repeated Amph exposures, changes of the transmitter content were correlated with reductions of 50-64% in the levels of the phosphorylated (p) MAP kinase (K) expression and 39-55% in the calbindin-D28k (CB) of the Purkinje cell somata, and increases of 289-556% in pCREB, 373-594% c-FOS, and 51-76% calretinin of the granular layer, as shown by immunohistochemical analysis. The up-regulated GABA content in the vermis and hemisphere may be associated with the decreased expression of Purkinje somatal CB and pMAPK, implicating a relation between the Ca(2+) of Purkinje cells and the level of GABA. Furthermore, the prominent increases of the granular layer pCREB, c-FOS and calretinin may influence the activity of Purkinje cells, which are known to be modulated by the granule cells. The data indicate that the Amph exposure selectively alters specific transmitters in the cerebellar lobes and modifies the cellular expression of distinct signaling proteins in the cerebellar layers.

Oxidative stress is thought to play an important role in the pathogenesis of Parkinson's disease (PD). Glutathione (GSH), a major cellular antioxidant, is decreased in the substantia nigra pars compacta of PD patients. The aim of the present study was to investigate whether deprenyl and its desmethyl metabolite, putative neuroprotective agents in the treatment of PD, could protect cultured rat mesencephalic neurons from cell death caused by GSH depletion due to treatment with L-buthionine-(S,R)-sulfoximine (BSO). BSO (10 microM) caused extensive cell death after 48 hr, as demonstrated by disruption of cellular integrity and release of lactate dehydrogenase into the culture medium. Both deprenyl and desmethylselegiline, at concentrations of 5 and 50 microM, significantly protected dopaminergic neurons from toxicity without preventing the BSO-induced loss in GSH. Protection was not associated with monoamine oxidase type B inhibition in that pargyline, a potent MAO inhibitor, was ineffective and pretreatment with pargyline did not prevent the protective effects of deprenyl. Protection was not associated with inhibition of dopamine uptake by deprenyl because the dopamine uptake inhibitor mazindol did not diminish BSO toxicity. The antioxidant ascorbic acid (200 microM) also protected against BSO-induced cell death, suggesting that oxidative events were involved. This study demonstrates that deprenyl and its desmethyl metabolite can diminish cell death associated with GSH depletion.

The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones. When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 microM) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in [3H]DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration. Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons. At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values. Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions. Ascorbate (200 microM), an antioxidant, prevented the rise in GSH. The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content. On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in [3H]DA uptake. The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity. In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, L-DOPA prevented cell death by L-BSO.