Efficacy of small-molecule glycogen synthase kinase-3 inhibitors in the postnatal rat model of tau hyperphosphorylation.

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimers disease (AD) such as hyperphosphorylation of the protein, tau. GSK-3beta expression, enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here, we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses, p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau, GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats, while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells, using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis.

Evidence for dysfunction of the nigrostriatal pathway in the R6/1 line of transgenic Huntington's disease mice.

The present multidisciplinary study examined nigrostriatal dopamine and striatal amino acid transmission in the R6/1 line of transgenic Huntington's disease (HD) mice expressing exon 1 of the HD gene with 115 CAG repeats. Although the number of tyrosine hydroxylase-positive neurons was not reduced and nigrostriatal connectivity remained intact in 16-week-old R6/1 mice, the size of tyrosine hydroxylase-positive neurons in the substantia nigra was reduced by 15%, and approximately 30% of these cells exhibited aggregated huntingtin. In addition, using in vivo microdialysis, we found that basal extracellular striatal dopamine levels were reduced by 70% in R6/1 mice compared to their wild-type littermates. Intrastriatal perfusion with malonate in R6/1 mice resulted in a short-lasting, attenuated increase in local dopamine release compared to wild-type mice. Furthermore, the size of the malonate-induced striatal lesion was 80% smaller in these animals. Taken together, these findings suggest that a functional deficit in nigrostriatal dopamine transmission may contribute to the behavioral phenotype and the resistance to malonate-induced neurotoxicity characteristic of R6/1 HD mice.

Fullerene-based antioxidants and neurodegenerative disorders.

Water-soluble derivatives of buckminsterfullerene (C(60)) derivatives are a unique class of compounds with potent antioxidant properties. Studies on one class of these compounds, the malonic acid C(60) derivatives (carboxyfullerenes), indicated that they are capable of eliminating both superoxide anion and H(2)O(2), and were effective inhibitors of lipid peroxidation, as well. Carboxyfullerenes demonstrated robust neuroprotection against excitotoxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesencephalic dopaminergic neurons from both MPP(+) and 6-hydroxydopamine-induced degeneration. Although there is limited in vivo data on these compounds to date, we have previously reported that systemic administration of the C(3) carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis (FALS). Ongoing studies in other animal models of CNS disease states suggest that these novel antioxidants are potential neuroprotective agents for other neurodegenerative disorders, including Parkinson's disease.

Role of mitochondrial dysfunction and dopamine-dependent oxidative stress in amphetamine-induced toxicity.

To define the molecular mechanisms underlying amphetamine (AMPH) neurotoxicity, primary cultures of dopaminergic neurons were examined for drug-induced changes in dopamine (DA) distribution, oxidative stress, protein damage, and cell death. As in earlier studies, AMPH rapidly redistributed vesicular DA to the cytoplasm, where it underwent outward transport through the DA transporter. DA was concurrently oxidized to produce a threefold increase in free radicals, as measured by the redox-sensitive dye dihydroethidium. Intracellular DA depletion using the DA synthesis inhibitor alpha-methyl-p-tyrosine or the vesicular monoamine transport blocker reserpine prevented drug-induced free radical formation. Despite these AMPH-induced changes, neither protein oxidation nor cell death was observed until 1 and 4 days, respectively. AMPH also induced an early burst of free radicals in a CNS-derived dopaminergic cell line. However, AMPH-mediated attenuation of ATP production and mitochondrial function was not observed in these cells until 48 to 72 hours. Thus, neither metabolic dysfunction nor loss of viability was a direct consequence of AMPH neurotoxicity. In contrast, when primary cultures of dopaminergic neurons were exposed to AMPH in the presence of subtoxic doses of the mitochondrial complex I inhibitor rotenone, cell death was dramatically increased, mimicking the effects of a known parkinsonism-inducing toxin. Thus, metabolic stress may predispose dopaminergic neurons to injury by free radical-promoting insults such as AMPH.

The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity.

Uptake of the Parkinsonism-inducing toxin, 1-methyl-4-phenylpyridinium (MPP(+)), into dopaminergic terminals is thought to block Complex I activity leading to ATP loss and overproduction of reactive oxygen species (ROS). The present study indicates that MPP(+)-induced ROS formation is not mitochondrial in origin but results from intracellular dopamine (DA) oxidation. Although a mean lethal dose of MPP(+) led to ROS production in identified dopaminergic neurons, toxic doses of the Complex I inhibitor rotenone did not. Concurrent with ROS formation, MPP(+) redistributed vesicular DA to the cytoplasm prior to its extrusion from the cell by reverse transport via the DA transporter. MPP(+)-induced DA redistribution was also associated with cell death. Depleting cells of newly synthesized and/or stored DA significantly attenuated both superoxide production and cell death, whereas enhancing intracellular DA content exacerbated dopaminergic sensitivity to MPP(+). Lastly, depleting cells of DA in the presence of succinate completely abolished MPP(+)-induced cell death. Thus, MPP(+) neurotoxicity is a multi-component process involving both mitochondrial dysfunction and ROS generated by vesicular DA displacement. These results suggest that in the presence of a Complex I defect, misregulation of DA storage could lead to the loss of nigrostriatal neurons in Parkinson's disease.

Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons.

Oxidative stress is thought to contribute to dopaminergic cell death in Parkinson's disease (PD). The neurotoxin 6-hydroxydopamine (6-OHDA), which is easily oxidized to reactive oxygen species (ROS), appears to induce neuronal death by a free radical-mediated mechanism, whereas the involvement of free radicals in N-methyl-4-phenylpyridinium (MPP+) toxicity is less clear. Using free radical-sensitive fluorophores and vital dyes with post hoc identification of tyrosine hydroxylase-positive neurons, we monitored markers of apoptosis and the production of ROS in dopaminergic neurons treated with either 6-OHDA or MPP+. Annexin-V staining suggested that 6-OHDA but not MPP+-mediated cell death was apoptotic. In accordance with this assignment, the general caspase inhibitor Boc-(Asp)-fluoromethylketone only blocked 6-OHDA neurotoxicity. Both toxins exhibited an early, sustained rise in ROS, although only 6-OHDA induced a collapse in mitochondrial membrane potential temporally related to the increase in ROS. Recently, derivatives of buckminsterfullerene (C60) molecules have been shown to act as potent antioxidants in several models of oxidative stress (Dugan et al., 1997). Significant, dose-dependent levels of protection were also seen in these in vitro models of PD using the C3 carboxyfullerene derivative. Specifically, C3 was fully protective in the 6-OHDA paradigm, whereas it only partially rescued dopaminergic neurons from MPP+-induced cell death. In either model, it was more effective than glial-derived neurotrophic factor. These data suggest that cell death in response to 6-OHDA and MPP+ may progress through different mechanisms, which can be partially or entirely saved by carboxyfullerenes.