RNA sequencing of olfactory bulb in Parkinson's disease reveals gene alterations associated with olfactory dysfunction.

The olfactory bulb is involved early in the pathophysiology of Parkinson's disease (PD), which is consistent with the early onset of olfactory dysfunction. Identifying the molecular mechanisms through which PD affects the olfactory bulb could lead to a better understanding of the pathophysiology and etiology of olfactory dysfunction in PD. We specifically aimed to assess gene expression changes, affected pathways and co-expression network by whole transcriptomic profiling of the olfactory bulb in subjects with clinicopathologically defined PD. Bulk RNA sequencing was performed on frozen human olfactory bulbs of 20 PD and 20 controls without dementia or any other neurodegenerative disorder, from the Arizona Study of Aging and Neurodegenerative disorders and the Brain and Body Donation Program. Differential expression analysis (19 PD vs 19 controls) revealed 2164 significantly differentially expressed genes (1090 upregulated and 1074 downregulated) in PD. Pathways enriched in downregulated genes included oxidative phosphorylation, olfactory transduction, metabolic pathways, and neurotransmitters synapses while immune and inflammatory responses as well as cellular death related pathways were enriched within upregulated genes. An overrepresentation of microglial and astrocyte-related genes was observed amongst upregulated genes, and excitatory neuron-related genes were overrepresented amongst downregulated genes. Co-expression network analysis revealed significant modules highly correlated with PD and olfactory dysfunction that were found to be involved in the MAPK signaling pathway, cytokine-cytokine receptor interaction, cholinergic synapse, and metabolic pathways. LAIR1 (leukocyte associated immunoglobulin like receptor 1) and PPARA (peroxisome proliferator activated receptor alpha) were identified as hub genes with a high discriminative power between PD and controls reinforcing an important role of neuroinflammation in the olfactory bulb of PD subjects. Olfactory identification test score positively correlated with expression of genes coding for G-coupled protein, glutamatergic, GABAergic, and cholinergic receptor proteins and negatively correlated with genes for proteins expressed in glial olfactory ensheathing cells. In conclusion, this study reveals gene alterations associated with neuroinflammation, neurotransmitter dysfunction, and disruptions of factors involved in the initiation of olfactory transduction signaling that may be involved in PD-related olfactory dysfunction.

Whole-Cell Dissociated Suspension Analysis in Human Brain Neurodegenerative Disease: A Pilot Study.

Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of Whole Cell Dissociated Suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and Fluorescence Activated Cell Sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis.