sirolimus and Parkinson-Disease--Secondary

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Topics: Adenine; Animals; Autophagy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Transgenic; MTOR Inhibitors; Neuroinflammatory Diseases; Neuroprotective Agents; Parkinson Disease; Parkinson Disease, Secondary; Signal Transduction; Sirolimus; Substantia Nigra; TOR Serine-Threonine Kinases; Trehalose

Cell-to-cell spreading of misfolded α-synuclein (α-syn) is suggested to contribute to the progression of neuropathology in Parkinson's disease (PD). Compelling evidence supports the hypothesis that misfolded α-syn transmits from neuron-to-neuron and seeds aggregation of the protein in the recipient cells. Furthermore, α-syn frequently appears to propagate in the brains of PD patients following a stereotypic pattern consistent with progressive spreading along anatomical pathways. We have generated a C. elegans model that mirrors this progression and allows us to monitor α-syn neuron-to-neuron transmission in a live animal over its lifespan. We found that modulation of autophagy or exo/endocytosis, affects α-syn transfer. Furthermore, we demonstrate that silencing C. elegans orthologs of PD-related genes also increases the accumulation of α-syn. This novel worm model is ideal for screening molecules and genes to identify those that modulate prion-like spreading of α-syn in order to target novel strategies for disease modification in PD and other synucleinopathies.

Topics: Adenosine Triphosphatases; Aldehyde Oxidoreductases; alpha-Synuclein; Animals; Autophagy; Brain; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Communication; Discoidin Domain Receptor 2; Disease Models, Animal; Endocytosis; Exocytosis; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Neurons; Parkinson Disease, Secondary; Protein Aggregates; Protein Serine-Threonine Kinases; Protein Transport; RNA, Small Interfering; Sirolimus; Spectrometry, Fluorescence; Tryptophan Hydroxylase; Ubiquitin-Protein Ligases

Topics: 1-Methyl-4-phenylpyridinium; Acids; Autophagy; Cathepsin D; Cell Death; Cell Line; Dopamine Agents; Humans; Lysosomes; Parkinson Disease, Secondary; Phagosomes; Sirolimus; Trehalose

In neurodegenerative disorders such as Parkinson's disease (PD), autophagy is implicated in the process of dopaminergic neuron cell death. The α-synuclein protein is a major component of Lewy bodies and Lewy neurites, and mutations in α-synuclein have been implicated in the etiology of familial PD. The current work investigates the mechanisms underlying the therapeutic effects of the autophagy-stimulating antibiotic rapamycin in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Male C57BL/6 mice were treated with intravenous rapamycin or saline control for 7 days following MPTP administration. Immunohistochemistry and western blotting were used to detect alterations in the expression of PD biomarkers, including tyrosine hydroxylase (TH), and the level of autophagy was evaluated by the detection of both microtubule-associated protein light chain 3 (LC3) and α-Synuclein cleavage. In addition, levels of monoamine neurotransmitters were measured in the striatum using high performance liquid chromatography (HPLC). Immunohistochemistry using antibodies against TH indicated that the number of dopaminergic neurons in the substantia nigra following MPTP treatment was significantly higher in rapamycin-treated mice compared with saline-treated controls (p < 0.01). Levels of TH expression in the striatum were similar between the groups. α-synuclein Immunoreactivity was significantly decreased in rapamycin-treated mice compared with controls (p < 0.01). Immunoreactivity for LC3, however, was significantly higher in the rapamycin-treated animals than controls (p < 0.01). The concentrations of both striatal dopamine, and the dopamine metabolite DOPAC, were significantly decreased in both MPTP-treated groups compared with untreated controls. The loss of DOPAC was less severe in rapamycin-treated mice compared with saline-treated mice (p < 0.01) following MPTP treatment. These results demonstrate that treatment with rapamycin is able to prevent the loss of TH-positive neurons and to ameliorate the loss of DOPAC following MPTP treatment, likely via activation of autophagy/lysosome pathways. Thus, further investigation into the effectiveness of rapamycin administration in the treatment of PD is warranted.

Topics: alpha-Synuclein; Animals; Anti-Bacterial Agents; Autophagy; Biogenic Monoamines; Biomarkers; Blotting, Western; Brain; Chromatography, High Pressure Liquid; Electrochemistry; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; MPTP Poisoning; Neostriatum; Neurotransmitter Agents; Parkinson Disease, Secondary; Sirolimus; Tyrosine 3-Monooxygenase

Recombinant AAV vectors containing a dimerizer-inducible system of transcriptional activation provide a strategy for control of therapeutic gene expression in the CNS. Here we explored this system for regulated expression of human aromatic L-amino acid decarboxylase (hAADC) in a rodent model of Parkinson disease. Expression of hAADC, the enzyme that converts L-dopa to dopamine, was dependent on reconstitution of a functional transcription factor (TF) by the dimerizer rapamycin. Two vectors, AAV-CMV-TF and AAV-Z12-hAADC, were infused into striata of 6-OHDA-lesioned rats. Rapamycin-induced increases in expression of hAADC repeatedly produced robust rotational behavior in response to low doses of L-dopa. Seven weeks after vector infusion, AADC expression in brain was quantitated by both stereology and Western blot analysis following the final rapamycin treatment. While a low level of hAADC was observed in rats that were not induced with rapamycin, this basal expression was not significant enough to elicit a rotational response to L-dopa. This study demonstrated a robust behavioral response of parkinsonian rats to regulated hAADC expression. Recombinant AAV vectors controlled by rapamycin or its analogs show promise as candidates for CNS therapies in which regulation of the transgene is desired.

Topics: Animals; Antiparkinson Agents; Aromatic-L-Amino-Acid Decarboxylases; Cell Line; Corpus Striatum; Dependovirus; Dimerization; Disease Models, Animal; Dopamine; Gene Dosage; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Humans; Levodopa; Oxidopamine; Parkinson Disease, Secondary; Rats; Rats, Sprague-Dawley; Recombination, Genetic; Sirolimus; Stereotyped Behavior; Transduction, Genetic