Studies have shown that ␣-synuclein (␣-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) ␣-syn and two human ␣-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant ␣-syn expressed on the injected side was about four times the endogenous rat ␣-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt ␣-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates ␣-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates ␣-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease.
Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.
Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.
AmBisome is a lyophilized preparation of liposomal amphotericin B. The acute intravenous toxicity of AmBisome was evaluated in mice and rats, and the LD50S were found to be greater than 175 and 50 mg/kg, respectively. The corresponding LD50S for conventional amphotericin B were approximately 2.3 and 1.6 mg/kg for mice and rats, respectively. The multiple dose toxicity test confirmed that AmBisome was well tolerated by both species. There were no deaths observed among mice receiving 25 or 50 mg/kg AmBisome for 14 days, and only two deaths among mice receiving 75 mg/kg AmBisome. One rat died in the group receiving 25 mg/kg AmBisome for 30 days. However, five of ten and nine of ten rats died in the groups treated with 50 and 75 mg/kg AmBisome, respectively. Hepatotoxicity was evident by elevation in serum liver enzyme levels for these groups. Initial pharmacokinetic evaluations demonstrated that peak plasma concentrations of 87 and 118 mg/kg, respectively, were attained in mice and rats after injection with 5 mg/kg AmBisome. Terminal plasma half-lives of 3.36 and 7.56 h were calculated for mice and rats, respectively. Tissue accumulations of amphotericin B resulting from multiple dose intravenous administration of either conventional amphotericin B or AmBisome were determined. At equivalent doses of 1 mg/kg, AmBisome treatment resulted in higher liver and spleen uptake of drug, but lower kidney and lung uptake than amphotericin B. At 5 mg/kg, AmBisome treatment resulted in concentrations of drug in the kidney and lungs that were comparable to corresponding tissue levels observed in the group treated with 1 mg/kg conventional amphotericin B.(ABSTRACT TRUNCATED AT 250 WORDS)
Intraventricular administration of glial cell line-derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostriatal tract passes through the lateral hypothalamus, motor hyperactivity mediated by nigrostriatal dopamine (DA) may have been responsible for the previously observed effect on body weight. In this study, we compared bilateral injections of rAAV2/5-GDNF in hypothalamus versus substantia nigra (SN) in aged Brown-Norway X Fisher 344 rats. Nigrostriatal GDNF overexpression resulted in significantly greater weight loss than rats treated in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to motor activity levels of the rats, though some of the weight loss could be attributed to a transient reduction in food intake. Forebrain DA levels did not account for the observed effects on body weight, although GDNF-induced increases in nucleus accumbens DA may have partially contributed to this effect in the hypothalamic GDNF-treated group. However, only nigrostriatal GDNF overexpression induced activation of phosphorylated extracellular signal-regulated kinase (p-ERK) in a small population of corticotrophin-releasing factor [corticotrophin-releasing hormone (CRH)] neurons located specifically in the medial parvocellullar division (MPD) of the paraventricular nucleus of the hypothalamus. Activation of these hypothalamic CRH neurons likely accounted for the observed metabolic effects leading to weight loss in obese rats.
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