Sirtuin 2 (SIRT2) is one of seven known mammalian protein deacetylases homologous to the yeast master lifespan regulator Sir2. In recent years, the sirtuin protein deacetylases have emerged as candidate therapeutic targets for many human diseases, including metabolic and age-dependent neurological disorders. In non-neuronal cells, SIRT2 has been shown to function as a tubulin deacetylase and a key regulator of cell division and differentiation. However, the distribution and function of the SIRT2 microtubule (MT) deacetylase in differentiated, postmitotic neurons remain largely unknown. Here, we show abundant and preferential expression of specific isoforms of SIRT2 in the mammalian central nervous system and find that a previously uncharacterized form, SIRT2.3, exhibits age-dependent accumulation in the mouse brain and spinal cord. Further, our studies reveal that focal areas of endogenous SIRT2 expression correlate with reduced α-tubulin acetylation in primary mouse cortical neurons and suggest that the brain-enriched species of SIRT2 may function as the predominant MT deacetylases in mature neurons. Recent reports have demonstrated an association between impaired tubulin acetyltransferase activity and neurodegenerative disease; viewed in this light, our results showing age-dependent accumulation of the SIRT2 neuronal MT deacetylase in wild-type mice suggest a functional link between tubulin acetylation patterns and the aging brain.

We applied single-cell RNA sequencing to profile genome-wide gene expression in about 9400 individual cerebellar cells from the mouse embryo at embryonic day 13.5. Reiterative clustering identified the major cerebellar cell types and subpopulations of different lineages. Through pseudotemporal ordering to reconstruct developmental trajectories, we identified novel transcriptional programs controlling cell fate specification of populations arising from the ventricular zone and the rhombic lip, two distinct germinal zones of the embryonic cerebellum. Together, our data revealed cell-specific markers for studying the cerebellum, gene-expression cascades underlying cell fate specification, and a number of previously unknown subpopulations that may play an integral role in the formation and function of the cerebellum. Our findings will facilitate new discovery by providing insights into the molecular and cell type diversity in the developing cerebellum.

SUMMARYWe applied single-cell RNA sequencing to profile genome-wide gene expression in about 9,400 individual cerebellar cells from the mouse embryo at embryonic day 13.5. Reiterative clustering identified the major cerebellar cell types and subpopulations of different lineages. Through pseudotemporal ordering to reconstruct developmental trajectories, we identified novel transcriptional programs controlling cell fate specification of populations arising from the ventricular zone and the anterior rhombic lip, two distinct germinal zones of the embryonic cerebellum. Together, our data revealed cell-specific markers for studying the cerebellum, important specification decisions, and a number of previously unknown subpopulations that may play an integral role in the formation and function of the cerebellum. Importantly, we identified a potential mechanism of vermis formation, which is affected by multiple congenital cerebellar defects. Our findings will facilitate new discovery by providing insights into the molecular and cell type diversity in the developing cerebellum.

Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p <0.05), whereas Pad2 levels did not change significantly (p >0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.