Mattei, D. | Search bioRxiv

bioRxiv2020

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37{degrees}C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4{degrees}C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

neuroscience↗

bioRxiv2020

A CRISPR-Cas9-engineered mouse model for GPI anchor deficiency mirrors human phenotype and shows hippocampal synaptic dysfunctions.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI) anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C>A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This new mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development. Significance statementInherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated the first viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using the new model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.

neuroscience↗