ATF6ß Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6ß, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6ß deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6ß deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6ß could play a role in psychiatric conditions in the emotional centers of the brain.

Inhibition of CD38 and supplementation of nicotinamide riboside ameliorate lipopolysaccharide-induced microglial and astrocytic neuroinflammation by increasing NAD.

Neuroinflammation is initiated by activation of the brain's innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in various neurological conditions including multiple sclerosis and Alzheimer's disease. Nicotinamide adenine dinucleotide (NAD+ ) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that deletion of CD38, which consumes NAD+ , suppressed cuprizone-induced demyelination, neuroinflammation, and glial activation. However, it is still unknown whether CD38 directly affects neuroinflammation through regulating brain NAD+ level. In this study, we investigated the effect of CD38 deletion and inhibition and supplementation of NAD+ on lipopolysaccharide (LPS)-induced neuroinflammation in mice. Intracerebroventricular injection of LPS significantly increased CD38 expression especially in the hippocampus. Deletion of CD38 decreased LPS-induced inflammatory responses and glial activation. Pre-administration of apigenin, a flavonoid with CD38 inhibitory activity, or nicotinamide riboside (NR), an NAD+ precursor, increased NAD+ level, and significantly suppressed induction of cytokines and chemokines, glial activation and subsequent neurodegeneration after LPS administration. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD+ , NR or 78c, the latter a specific CD38 inhibitor. Finally, all these compounds suppressed NF-κB signaling pathway in microglia. These results suggest that CD38-mediated neuroinflammation is linked to NAD+ consumption and that boosting NAD+ by CD38 inhibition and NR supplementation directly suppress neuroinflammation in the brain.

Deletion of CD38 and supplementation of NAD+ attenuate axon degeneration in a mouse facial nerve axotomy model.

Following facial nerve axotomy, nerve function is not fully restored even after reconstruction. This may be attributed to axon degeneration/neuronal death and sustained neuroinflammation. CD38 is an enzyme that catalyses the hydrolysis of nicotinamide adenine dinucleotide (NAD+) and is a candidate molecule for regulating neurodegeneration and neuroinflammation. In this study, we analyzed the effect of CD38 deletion and NAD+ supplementation on neuronal death and glial activation in the facial nucleus in the brain stem, and on axon degeneration and immune cell infiltration in the distal portion of the facial nerve after axotomy in mice. Compared with wild-type mice, CD38 knockout (KO) mice showed reduced microglial activation in the facial nucleus, whereas the levels of neuronal death were not significantly different. In contrast, the axon degeneration and demyelination were delayed, and macrophage accumulation was reduced in the facial nerve of CD38 KO mice after axotomy. Supplementation of NAD+ with nicotinamide riboside slowed the axon degeneration and demyelination, although it did not alter the level of macrophage infiltration after axotomy. These results suggest that CD38 deletion and supplementation of NAD+ may protect transected axon cell-autonomously after facial nerve axotomy.

A monoclonal antibody raised against a synthetic oxytocin peptide stains mouse hypothalamic neurones.

A monoclonal antibody against oxytocin was generated in 7a5 hybridoma cells derived from myeloma cells and lymphocytes from the spleen of mice immunised with a synthetic oxytocin peptide. The 7a5 monoclonal antibody bound with oxytocin in enzyme-linked immunosorbent assays. 7a5 cell growth medium was diluted up to 5000-fold and used for immunohistochemistry. First, to test the specificity of the 7a5 antibody against oxytocin, we stained brain tissues of oxytocin knockout mice, comprising mice in which the first exon of the oxytocin-neurophysin gene is deleted. No 7a5 immunoreactivity was detected in the paraventricular nucleus (PVN) of the hypothalamus of oxytocin knockout mice; however, this area was strongly stained with the anti-vasopressin polyclonal antibody, HM07. Tissue preparations of the wild-type mouse PVN and supraoptic nucleus (SON) displayed 7a5 immunoreactivity that was indistinguishable from the staining produced with an anti-oxytocin polyclonal antibody, HM06. The immunoreactivity of HM06 in the PVN was similar to that of an anti-oxytocin monoclonal antibody, PS38. We then examined the cross-reactivity of 7a5 with arginine vasopressin. The majority of cell soma and processes stained by 7a5 were not co-stained with the vasopressin antibody in SON and PVN regions. Furthermore, the suprachiasmatic nucleus was stained by the vasopressin antibody but not by 7a5. These results demonstrate that 7a5 is a new anti-oxytocin monoclonal antibody recognising oxytocin and not vasopressin; therefore, 7a5 can be used to investigate the role of oxytocin in the brain.

DBZ (DISC1-binding zinc finger protein)-deficient mice display abnormalities in basket cells in the somatosensory cortices.

Disrupted-in-schizophrenia 1 (DISC1)-binding zinc finger protein (DBZ) is a DISC1-interacting molecule and the interaction between DBZ and DISC1 is involved in neurite outgrowth in vitro. DBZ is highly expressed in brain, especially in the cortex. However, the physiological roles of DBZ in vivo have not been clarified. Here, we show that development of basket cells, a morphologically defined class of parvalbumin (PV)-containing interneurons, is disturbed in DBZ knockout (KO) mice. DBZ mRNA was highly expressed in the ventral area of the subventricular zone of the medial ganglionic eminence, where PV-containing cortical interneurons were generated, at embryonic 14.5 days (E14.5). Although the expression level for PV and the number of PV-containing interneurons were not altered in the cortices of DBZ KO mice, basket cells were less branched and had shorter processes in the somatosensory cortices of DBZ KO mice compared with those in the cortices of WT mice. Furthermore, in the somatosensory cortices of DBZ KO mice, the level of mRNAs for the gamma-aminobutyric acid-synthesizing enzymes GAD67 was decreased. These findings show that DBZ is involved in the morphogenesis of basket cells.

Long-term inhibition of Rho-kinase suppresses neointimal formation after stent implantation in porcine coronary arteries: involvement of multiple mechanisms.

OBJECTIVE: We recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is substantially involved in the pathogenesis of arteriosclerosis. In this study, we examined whether Rho-kinase is also involved in in-stent restenosis and if so, what mechanism is involved. METHODS AND RESULTS: Pigs underwent stent implantation in the left coronary artery with or without administration of fasudil (30 mg/kg per day orally), a specific Rho-kinase inhibitor, starting 2 days before the procedure for a duration of 4 weeks. On day 28, reductions in coronary diameter and neointimal formation associated with macrophage accumulation, collagen deposition, and transforming growth factor (TGF)-beta1 expression were noted at the stent site, and all were significantly suppressed by fasudil. On day 7, fasudil significantly increased the frequency of TUNEL-positive apoptotic cells, while it tended to reduce that of bromodeoxyuridine-positive proliferating cells in the neointima. Western blot analysis on day 7 demonstrated that phosphorylations of the ezrin/radixin/moesin family (a marker of Rho-kinase activity in vivo) and protein expression of monocyte chemoattractant protein-1and bcl-2 were upregulated at the stent site and were significantly suppressed by fasudil. CONCLUSIONS: These results indicate that long-term inhibition of Rho-kinase suppresses in-stent neointimal formation by multiple mechanisms, including reduced vascular inflammation, enhanced apoptosis, and decreased collagen deposition.