Minimizing the ex vivo confounds of cell-isolation techniques on transcriptomic and translatomic profiles of purified microglia Abbreviated Title (50 character maximum):Microglial activation during cell isolation 3. List all Author Names and Affiliations in order as they would appear in the

Elongation of very long chain fatty acids-4 (ELOVL4) is essential for synthesis of very long chain polyunsaturated and saturated fatty acids (VLC-PUFA and VLC-SFA, respectively) of chain length greater than 26 carbons. Mutations in the ELOVL4 gene cause several distinct neurodegenerative diseases including Stargardt-like macular dystrophy (STGD3), spinocerebellar ataxia 34 (SCA34), and a neuro-ichthyotic syndrome with severe seizures and spasticity, as well as erythrokeratitis variabilis (EKV), a skin disorder. However, the relationship between ELOVL4 mutations, its VLC-PUFA and VLC-SFA products, and specific neurological symptoms remains unclear. We generated a knock-in rat line (SCA34-KI) that expresses the 736T>G (p.W246G) form of ELOVL4 that causes human SCA34. Lipids were analyzed by gas chromatography and mass spectrometry. Retinal function was assessed using electroretinography. Retinal integrity was assessed by histology, optical coherence tomography, and immunolabeling. Analysis of retina and skin lipids showed that the W246G mutation selectively impaired synthesis of VLC-SFA, but not VLC-PUFA. Homozygous SCA34-KI rats showed reduced ERG a- and b-wave amplitudes by 90 days of age, particularly for scotopic responses. Anatomical analyses revealed no indication of neurodegeneration in heterozygote or homozygote SCA34-KI rats out to 6–7 months of age. These studies reveal a previously unrecognized role for VLC-SFA in regulating retinal function, particularly transmission from photoreceptors to the inner retina, in the absence of neurodegeneration. Furthermore, these findings suggest that the tissue specificity and symptoms associated with disease-causing ELOVL4 mutations likely arise from selective differences in the ability of the mutant ELOVL4 enzymes to support synthesis of VLC-PUFA and/or VLC-SFA.

Background Microglia, the brain’s principal immune cells, have been implicated in the pathogenesis of Alzheimer’s disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. Methods Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5–6 month-old [mo]) and old (22–25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. Results There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally and autosomally encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface (https://neuroepigenomics.omrf.org/). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. Conclusions These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

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