Somatic copy number variant load in neurons of healthy controls and Alzheimer's disease patients.

The possible role of somatic copy number variations (CNVs) in Alzheimer's disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons.

Functional Role of γ-Crystallin N in the Auditory Hindbrain.

γ-crystallins are major components of the vertebrate lens but show expression in other tissues as well. Their extralenticular functions remain so far unclear. Here, we explored such roles in the rodent superior olivary complex in which previous analysis demonstrated developmentally regulated expression of Crygd, Cryge and Crygn. Immunohistochemistry with novel antibodies against Crygd/e and Crygn indicate that expression of Crygd/e was moderate and varied between the perinatal superior olivary complex of mice, rats, and gerbils. Crygn-immunoreactivity was more robust and consistently highest in the medial nucleus of the trapezoid body, but also present in other nuclei of the superior olivary complex. To analyze the function of Crygn in the auditory hindbrain, we used a Crygn allele with a floxed exon 2. Upon pairing with Egr2::Cre mice, exon 2, encoding the first two greek key motifs of Crygn, was deleted in the developing auditory hindbrain. Anatomical analysis of these mice revealed a 20% volume reduction in the medial nucleus of the trapezoid body and a 7% reduction in the lateral superior olive at postnatal day 25. This was due to cell loss between postnatal days 4 and 25, whereas cell size was unaffected. Auditory brainstem responses showed normal threshold but a significant increase in the amplitude of wave IV. Crygn is hence required for postmigratory survival and proper function of auditory hindbrain neurons. These results ascertain for the first time an essential extralenticular role for γ-crystallins in vivo.