As the second most frequent neurodegenerative disorder of the aging population, Parkinsons disease (PD) is characterized by progressive deficits in spontaneous movement, atrophy of dopaminergic midbrain neurons and aggregation of the protein alpha-synuclein (SNCA). regions on chromosome 4 (and loci) that contain genetic variants predisposing to multifactorial PD . Variations in the gene 3-untranslated region (3-UTR) and its promoter correlated strongly with PD risk . lpha-synuclein is physiologically concentrated in axon terminals. It is associated with the lipid membranes of synaptic vesicles and interacts with synaptobrevin, a component of the SNARE complex, mediating vesicle exocytosis and neurotransmitter release . Its toxic gain-of-function leads over time to impaired synaptic vesicle release and synaptic failure [9, 10]. Current investigations aim to elucidate alpha-synuclein-triggered pathology, concentrating on disease stages before the occurrence of irreversible cell loss, when neuroprotective therapies might still be efficacious. Here, we focused on two independent mouse lines of inbred FVB/N background with ~1.5-fold overexpression of human A53T-alpha-synuclein in nigrostriatal dopaminergic neurons under control of the heterologous neuron-specific prion-promoter. A53T-alpha-synuclein overexpression in these mice occurs in presynaptic nigral dopaminergic neurons and presynaptic cortical glutamatergic neurons, but not in postsynaptic striatal neurons. These mice display apparently normal movements at age 6?months, but progress to significantly impaired spontaneous locomotion by age 18?months, despite the absence of neuronal loss in the nigrostriatal projection . Previous expression profiling in these mice identified a transcript dysregulation throughout the brain and a 14-3-3 epsilon protein upregulation selectively in the striatum as molecular effects of alpha-synuclein triggered pathology. The alterations in these signalling molecules were temporally correlated with reduced striatal dopamine release and deficient long-term depression [12C14, 9]. To gain insight into the mechanisms underlying the impairment in vesicle exocytosis and neurotransmitter release, we surveyed progressive expression changes in midbrain/brainstem tissue using genome-wide unbiased transcriptome profiling. Promising candidates were validated with quantitative immunoblots. Results Overexpression of A53T-Alpha-Synuclein Modulates and mRNA Levels in Mouse Midbrain/Brainstem Previously documented (GEO database Manidipine dihydrochloride IC50 PIK3C2G “type”:”entrez-geo”,”attrs”:”text”:”GSE20547″,”term_id”:”20547″GSE20547, see also ) global transcriptome data from striatum, midbrain/brainstem and cerebellum of human A53T-alpha-synuclein overexpressing mice were filtered. We selected those significant changes at age 18?months relative to age 6?months, which were midbrain/brainstem-specific and were consistent between both transgenic mouse lines (PrPmtA and PrPmtB). Further selection prioritized those transcripts with no corresponding significant changes in wild-type midbrain/brainstem and in wild-type/transgenic striatum and cerebellum, resulting in the identification of 49 candidate effects of synucleinopathy (Table?1). Among the progressive upregulation effects, the increase of mRNA levels by A53T-alpha-synuclein overexpression was particularly interesting in view of our previous finding that (encoding forkhead box P1) is downregulated in alpha-synuclein knockout mouse . Thus, the midbrain-identity-mediating transcription factor Foxp1 appears to depend in its brain levels both on the gain-of-function and the loss-of-function of alpha-synuclein. Among the progressive downregulation effects, the decreased levels of (encoding complexin-1) selectively in the mutant midbrain/brainstem were especially interesting, in view of the co-localization of alpha-synuclein and complexin-1 at the SNARE complex. Other midbrain/brainstem-selective significant dysregulations of vesicle endocytosis/exocytosis pathway factors included the downregulation of transcript and the upregulation of mRNA expression in independent tissues by qPCR confirmed the significant downregulation in midbrain/brainstem of PrPmtA mice (Fig.?1). Table 1 Global transcriptome analysis of mice with nigrostriatal overexpression of human A53T-alpha-synuclein showing significant changes from Manidipine dihydrochloride IC50 age 6 to 18+?months Fig. 1 Quantitative real-time reverse transcriptase PCR demonstrates reduced mRNA levels of complexin-1 in the midbrain/brainstem of mice with A53T-alpha-synuclein overexpression. Tbp was always used as loading control, and mRNA level ratios were normalized … Overexpression of A53T-Alpha-Synuclein Leads to Elevated Complexin-1 Protein Levels in Mouse Midbrain/Brainstem Manidipine dihydrochloride IC50 We focused on the downregulation of midbrain/brainstem mRNA as a novel and promising effect, since the encoded protein complexin-1 is involved in the stimulus-dependent control of secretory vesicle exocytosis through the SNARE complex [21, 22]. Alpha-synuclein was also shown to modulate SNARE assembly and vesicle clustering, so this expression effect might constitute a very direct and early consequence of alpha-synuclein mutations. Densitometric analysis of immunoblots revealed a significant increase of complexin-1 protein levels in the midbrain/brainstem of aged A53T-alpha-synuclein overexpressing mice (Fig.?2aCc), despite mRNA downregulation. The alterations were readily apparent by ECL detection of membranes, making more sophisticated approaches such as near-infrared immunoblot detection or quantification by ELISA unnecessary. Fig. 2 Manidipine dihydrochloride IC50 Quantitative immunoblots demonstrate dysregulated levels of complexin-1 and 14-3-3epsilon proteins in the midbrain/brainstem of mice with alpha-synuclein mutation. Beta-actin was always used as loading control, and protein level ratios.