Single‐cell transcriptomic and functional characterization of cortical parvalbumin interneurons in a novel conditional knock‐out mouse lacking CSPα/DNAJC5

Abstract

Neurodegenerative diseases are progressive disorders currently without cure. Indeed, the molecular mechanisms underlying synaptic and neuronal degeneration are poorly understood. Cysteine String Protein‐α/DNAJC5 (CSPα/DNAJC5) is a synaptic co‐chaperone associated to neurodegeneration in humans. Adult‐onset autosomal dominant neuronal ceroid lipofuscinosis is caused by mutations in the gene DNAJC5. In mice, the genetic removal of CSPα/DNAJC5 leads to activity‐dependent synaptic degeneration, specially evident in the fast‐spiking GABAergic interneurons that express parvalbumin (PV). The early death of these mice limits the investigation of the natural history of the synaptic and neuronal dysfunction of these cells. We have now generated a conditional KO mouse (PVcre:Ai27D:Dnajc5flox/‐) lacking CSPα/DNAJC5 specifically in parvalbumin‐positive GABAergic interneurons labeled with the optogenetic actuator channelrhodopsin‐2 fused to the fluorescent reporter tdTomato. These mice develop a progressive neurological phenotype characterized by hyperactivity and ataxia without early lethality. At the neocortex, those neurons form disorganized perisomatic synapses with a reduced number of puncta, that, unexpectedly do not compromise the number of PV somata up to 8 months of age. Those neurons are, however, more reluctant to initiate high frequency action potential trains compared to controls. The spontaneous release of GABA from those neurons is altered. The frequency of mIPSCs is decreased, consistent with a lower number of functional synapses. The amplitude of mIPSCs is strikingly reduced and the kinetics modified, perhaps caused by alterations in vesicular GABA content and/or postsynaptic changes in GABA receptors. Further characterization of ion currents and pharmacological and optogenetic dissection of synaptic currents is required to understand the changes in excitability and synaptic transmission. In order to get insight into the homeostatic mechanisms of gene expression associated to synaptic dysfunction in PV interneurons lacking CSPα/DNAJC5, we have carried out the analysis of single cell transcriptomes of tdTomato+ cells isolated from the whole cortex of three different genotypes: PVcre:Ai27D:Dnajc5WT, PVcre:Ai27D:Dnajc5flox/+ and PVcre:Ai27D:Dnajc5flox/‐. In collaboration with Dr. Hjerling‐ Leffler's group (Karolinska Institute), we have combined fluorescence‐activated cell sorting (FACS), the STRT‐seq‐2i method and the WaferGen 9600‐well platform as a novel and powerful strategy for single‐cell RNA sequencing. Next, we have conducted computational analysis using the R toolkit Seurat. We have integrated scRNA‐seq data from the three genotypes (2847 cells) and, based on common sources of variation, we have identified 8 different populations (also known as identities) of PV interneurons and carried out a downstream comparative analysis of gene expression. Three of our PV identities were highly similar to PV subclasses recently described in specific cortical areas and one of them corresponded to chandelier cells. The gene ontology (GO) study based on the differential gene expression analysis suggests genetic dysregulation of metabolism and synaptic function, among other processes, in PVcre:Ai27D:Dnajc5flox/‐ neurons. Amid a wide repertoire of expression changes, our study has identified genes related to brain disorders and to the control of hyperexcitability. Those presumptive changes, however, still require validation with alternative methodologies before they can be used to explain the molecular mechanisms underlying the neuronal and the in vivo phenotypes found in the mice. Overall, the results presented in this thesis throw light and open multiple new perspectives to advance the understanding of molecular and circuit mechanisms underlying synaptic alterations of PV interneurons and the role of CSPα/DNAJC5 in brain disorders.