Propionic acid and fasudil as treatment against rotenone toxicity in an in vitro model of Parkinson's disease

Parkinson's disease (PD) is a multifactorial neurodegenerative disease. In recent years, several studies demonstrated that the gastroenteric system and intestinal microbiome influence central nervous system function. The pathological mechanisms triggered thereby change neuronal function in neurodegenerative diseases including dopaminergic neurons in Parkinson's disease. In this study, we employed a model system for PD of cultured primary mesencephalic cells and used the pesticide rotenone to model dopaminergic cell damage. We examined neuroprotective effects of the Rho kinase inhibitor Fasudil and the short chain fatty acid (SCFA) propionic acid on primary neurons in cell morphological assays, cell survival, gene and protein expression. Fasudil application resulted in significantly enhanced neuritic outgrowth and increased cell survival of dopaminergic cells. The application of propionic acid primarily promoted cell survival of dopaminergic cells against rotenone toxicity and increased neurite outgrowth to a moderate extent. Interestingly, Fasudil augmented gene expression of synaptophysin whereas gene expression levels of tyrosine hydroxylase (TH) were substantially increased by propionic acid. Concerning protein expression propionic acid treatment increased STAT3 levels but did not lead to an increased phosphorylation indicative of pathway activation. Our findings indicate that both Fasudil and propionic acid treatment show beneficial potential in rotenone-lesioned primary mesencephalic cells

A propagated skeleton approach to high throughput screening of neurite outgrowth for in vitro Parkinson's disease modelling

Neuronal models of neurodegenerative diseases such as Parkinson’s Disease (PD) are extensively studied in pathological and therapeutical research with neurite outgrowth being a core feature. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. In this study, we describe an autonomous computational assay for a high throughput skeletonization approach allowing for quantification of neurite outgrowth in large data sets from fluorescence microscopic imaging. Development and validation of the assay was conducted with differentiated SH-SY5Y cells and primary mesencephalic dopaminergic neurons (MDN) treated with the neurotoxic lesioning compound Rotenone. Results of manual annotation using NeuronJ and automated data were shown to correlate strongly ($R^{2}$-value 0.9077 for SH-SY5Y cells and $R^{2}$-value 0.9297 for MDN). Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula ($\it y$ = 0.5410 ⋅ $\it x$ + 1792; $\it y$ = 0.8789 ⋅ $\it x$ + 0.09191 for normalized results) with $\it y$ depicting automated and $\it x$ depicting manual data. This automated neurite length algorithm constitutes a valuable tool for modelling of neurite outgrowth that can be easily applied to evaluate therapeutic compounds with high throughput approaches

Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging

Bioluminescence imaging is a sensitive approach for longitudinal neuroimaging. Transgenic mice expressing luciferase under the promoter of doublecortin (DCX-luc), a specific marker of neuronal progenitor cells (NPC), allow monitoring of neurogenesis in living mice. Since the extent and time course of neurogenesis during autoimmune brain inflammation are controversial, we investigated neurogenesis in MOG-peptide induced experimental allergic encephalomyelitis (EAE) using DCX-luc reporter mice. We observed a marked, 2- to 4-fold increase of the bioluminescence signal intensity 10 days after EAE induction and a gradual decline 1–2 weeks thereafter. In contrast, immunostaining for DCX revealed no differences between EAE and control mice 2 and 4 weeks after immunization in zones of adult murine neurogenesis such as the dentate gyrus. $\textit {Ex vivo}$ bioluminescence imaging showed similar luciferase expression in brain homogenates of EAE and control animals. Apart from complete immunization including MOG-peptide also incomplete immunization with complete Freund´s adjuvant and pertussis toxin resulted in a rapid increase of the $\textit {in vivo}$ bioluminescence signal. Blood-brain barrier (BBB) leakage was demonstrated 10 days after both complete and incomplete immunization and might explain the increased bioluminescence signal $\textit {in vivo}$. We conclude, that acute autoimmune inflammation in EAE does not alter neurogenesis, at least at the stage of DCX-expressing NPC. Effects of immunization on the BBB integrity must be considered when luciferase is used as a reporter within the CNS during the active stage of EAE. Models with stable CNS-restricted luciferase expression could serve as technically convenient way to evaluate BBB integrity in a longitudinal manner