Concentration of non-myocyte proteins in arterial media of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

The most common inherited cause of vascular dementia and stroke, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is caused by mutations in NOTCH3. Post-translationally altered NOTCH3 accumulates in the vascular media of CADASIL arteries in areas of the vessels that exhibit profound cellular degeneration. The identification of molecules that concentrate in the same location as pathological NOTCH3 may shed light on processes that drive cytopathology in CADASIL. We performed a two phase immunohistochemical screen of markers identified in the Human Protein Atlas to identify new proteins that accumulate in the vascular media in a pattern similar to pathological NOTCH3. In phase one, none of 16 smooth muscle cell (SMC) localized antigens exhibited NOTCH3-like patterns of expression; however, several exhibited disease-dependent patterns of expression, with antibodies directed against FAM124A, GZMM, MTFR1, and ST6GAL demonstrating higher expression in controls than CADASIL. In contrast, in phase two of the study that included 56 non-SMC markers, two proteins, CD63 and CTSH, localized to the same regions as pathological NOTCH3, which was verified by VesSeg, a customized algorithm that assigns relative location of antigens within the layers of the vessel. Proximity ligation assays support complex formation between NOTCH3 fragments and CD63 in degenerating CADASIL media. Interestingly, in normal mouse brain, the two novel CADASIL markers, CD63 and CTSH, are expressed in non-SMC vascular cells. The identification of new proteins that concentrate in CADASIL vascular media demonstrates the utility of querying publicly available protein databases in specific neurological diseases and uncovers unexpected, non-SMC origins of pathological antigens in small vessel disease.

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice.

Background: The suprachiasmatic nucleus (SCN) within the hypothalamus is a key brain structure required to relay light information to the body and synchronize cell and tissue level rhythms and hormone release. Specific subpopulations of SCN neurons, defined by their peptide expression, regulate defined SCN output. Here we focus on the vasoactive intestinal peptide (VIP) expressing neurons of the SCN. SCN VIP neurons are known to regulate circadian rhythms and reproductive function. Methods: To specifically study SCN VIP neurons, we generated a novel knock out mouse line by conditionally deleting the SCN enriched transcription factor, Ventral Anterior Homeobox 1 (Vax1), in VIP neurons (Vax1Vip; Vax1fl/fl:VipCre). Results: We found that Vax1Vip females presented with lengthened estrous cycles, reduced circulating estrogen, and increased depressive-like behavior. Further, Vax1Vip males and females presented with a shortened circadian period in locomotor activity and ex vivo SCN circadian period. On a molecular level, the shortening of the SCN period was driven, at least partially, by a direct regulatory role of VAX1 on the circadian clock genes Bmal1 and Per2. Interestingly, Vax1Vip females presented with increased expression of arginine vasopressin (Avp) in the paraventricular nucleus, which resulted in increased circulating corticosterone. SCN VIP and AVP neurons regulate the reproductive gonadotropin-releasing hormone (GnRH) and kisspeptin neurons. To determine how the reproductive neuroendocrine network was impacted in Vax1Vip mice, we assessed GnRH sensitivity to a kisspeptin challenge in vivo. We found that GnRH neurons in Vax1Vip females, but not males, had an increased sensitivity to kisspeptin, leading to increased luteinizing hormone release. Interestingly, Vax1Vip males showed a small, but significant increase in total sperm and a modest delay in pubertal onset. Both male and female Vax1Vip mice were fertile and generated litters comparable in size and frequency to controls. Conclusion: Together, these data identify VAX1 in SCN VIP neurons as a neurological overlap between circadian timekeeping, female reproduction, and depressive-like symptoms in mice, and provide novel insight into the role of SCN VIP neurons.