Fluorescent Activated Nuclei Sorting (FANS) and a novel combinatorial fluidic indexing to elucidate molecular pathways involved in the pathogenesis in Parkinson’s Disease (PD)

MARTINO AVALLONE; JOAQUIN PARDO; SARA PALO; ULRICH PFISTERER; PER BRATTÅS ; JANA RAJOVA; MALIN ÅKERBLOM; MARCUS DAVIDSSON; TOMAS BJÖRKLUND

Paris

Congreso; Federation of European Neuroscience Societies 2022; 2022

Federation of European Neuroscience Societies

PD is a neurodegenerative disorder characterized by loss of dopamine (DA) neurons of the Substantia Nigra (SN). There is a big body of evidence suggesting that alpha synuclein (aSyn) plays a central role in the pathogenesis of PD. However, there is no molecularly precise, single cell in vivo transcriptomic studies with accurately defined aSyn overload. Here we describe a novel strategy and recent results based on AAVs engineering and transgenic animals. The MNM008 AAVs capsid, capable of retrograde transport in DA neurons, is injected in the striatum of TH-Cre rats and DAT-Cre mouse. The vector carries uniquely barcoded aSyn genomes or barcoded-nonpathogenic tagBFP.Injecting in the striatum with Cre-recombinase restriction of the expression to DA neurons of the SN, we avoid possible injection-related inflammation and cell loss. The nuclei from the micro dissected SN area are enriched for DA neurons using FANS. This is achieved thanks to the viral vector prep containing a Cre-inducible histone-linked GFP (H2B-GFP), which is only expressed in the population of interest. Using a combinatorial-indexing based single nuclei sequencing approach called Sci-Fi-Seq we can pre label all mRNA in each nucleus with an identifier for that specific sample. These samples are then pooled together and prepared for sequencing using 10X genomic. Immunohistochemistry results showed that the virus injected in the striatum was retrogradely transported and expressed in DA neurons of the SNpc. The FANS enrichment strategy for our population of interest expressing the virus, showed to be robust with an average of 2500 GFP positive (DA) nuclei retrieved per animal. Transcriptomic analysis on the GFP positive sorted nuclei revealed DA markers specific to the SN population, and the viral vector expression was restricted to this population, confirming the high selectivity of our approach.The combinatorial indexing and molecular barcoding will allow us to accurately measure the aSyn expression level in each DA neuron arranging them into disease stages in what we call pseudo-time. Moreover, this approach will identify causative cascades of changes at the transcriptional level. From this study we will further our knowledge on PD disease progression as well as possible new treatments.