Postnatal expression of CD38 in astrocytes regulates synapse formation and adult social memory.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

Isolation of ferret astrocytes reveals their morphological, transcriptional, and functional differences from mouse astrocytes.

Astrocytes play key roles in supporting the central nervous system structure, regulating synaptic functions, and maintaining brain homeostasis. The number of astrocytes in the cerebrum has markedly increased through evolution. However, the manner by which astrocytes change their features during evolution remains unknown. Compared with the rodent brain, the brain of the ferret, a carnivorous animal, has a folded cerebral cortex and higher white to gray matter ratio, which are common features of the human brain. To further clarify the features of ferret astrocytes, we isolated astrocytes from ferret neonatal brains, cultured these cells, and compared their morphology, gene expression, calcium response, and proliferating ability with those of mouse astrocytes. The morphology of cultured ferret astrocytes differed from that of mouse astrocytes. Ferret astrocytes had longer and more branched processes, smaller cell bodies, and different calcium responses to glutamate, as well as had a greater ability to proliferate, compared to mouse astrocytes. RNA sequencing analysis revealed novel ferret astrocyte-specific genes, including several genes that were the same as those in humans. Astrocytes in the ferret brains had larger cell size, longer primary processes in larger numbers, and a higher proliferation rate compared to mouse astrocytes. Our study shows that cultured ferret astrocytes have different features from rodent astrocytes and similar features to human astrocytes, suggesting that they are useful in studying the roles of astrocytes in brain evolution and cognitive functions in higher animals.

Localized astrogenesis regulates gyrification of the cerebral cortex.

The development and evolution of mammalian higher cognition are represented by gyrification of the laminar cerebral cortex and astrocyte development, but their mechanisms and interrelationships remain unknown. Here, we show that localized astrogenesis plays an important role in gyri formation in the gyrencephalic cerebral cortex. In functional genetic experiments, we show that reducing astrocyte number prevents gyri formation in the ferret cortex, while increasing astrocyte number in mice, which do not have cortical folds, can induce gyrus-like protrusions. Morphometric analyses demonstrate that the vertical expansion of deep pallial regions achieved by localized astrogenesis is crucial for gyri formation. Furthermore, our findings suggest that localized astrogenesis by a positive feedback loop of FGF signaling is an important mechanism underlying cortical folding in gyrencephalic mammalian brains. Our findings reveal both the cellular mechanisms and the mechanical principle of gyrification in the mammalian brain.

The ATF6ß-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity.

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of its paralogue ATF6ß remains elusive, especially in the central nervous system (CNS). Here, we demonstrate that ATF6ß is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin (CRT), a molecular chaperone in the ER with a high Ca2+-binding capacity. CRT expression was reduced to ~ 50% in the CNS of Atf6b-/- mice under both normal and ER stress conditions. Analysis using cultured hippocampal neurons revealed that ATF6ß deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death. The higher levels of death in Atf6b-/- neurons were recovered by ATF6ß and CRT overexpressions, or by treatment with Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and with an ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in the hippocampi of Atf6b-/- and Calr+/- mice, and restored by administration of 2-APB and salubrinal. These results suggest that the ATF6ß-CRT axis promotes neuronal survival under ER stress and excitotoxity by improving intracellular Ca2+ homeostasis.

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.

Abnormal social behavior and altered gene expression in mice lacking NDRG2.

N-myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family, has multiple functions in cell proliferation, differentiation, and stress responses, and is predominantly expressed by astrocytes in the central nervous system. Previous studies including ours demonstrated that NDRG2 is involved in various central nervous system pathologies. However, the significance of NDRG2 in neurodevelopment is not fully understood. Here, we investigated the expression profile of NDRG2 during postnatal brain development, the role of NDRG2 in social behavior, and transcriptome changes in the brain of NDRG2-deficient mice. NDRG2 expression in the brain increased over time from postnatal day 1 to adulthood. Deletion of NDRG2 resulted in abnormal social behavior, as indicated by reduced exploratory activity toward a novel mouse in a three-chamber social interaction test. Microarray analysis identified genes differentially expressed in the NDRG2-deficient brain, and upregulated gene expression of Bmp4 and Per2 was confirmed by quantitative PCR analysis. Expression of both these genes and the encoded proteins increased over time during postnatal brain development, similar to NDRG2. Gene expression of Bmp4 and Per2 was upregulated in cultured astrocytes isolated from NDRG2-deficient mice. These results suggest that NDRG2 contributes to brain development required for proper social behavior by modulating gene expression in astrocytes.

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.

Deletion of CD38 Suppresses Glial Activation and Neuroinflammation in a Mouse Model of Demyelination.

CD38 is an enzyme that catalyzes the synthesis of cyclic adenosine diphosphate-ribose from nicotinamide adenine dinucleotide (NAD+). We recently reported that this molecule regulates the maturation and differentiation of glial cells such as astrocytes and oligodendrocytes (OLs) in the developing brain. To analyze its role in the demyelinating situation, we employed cuprizone (CPZ)-induced demyelination model in mice, which is characterized by oligodendrocyte-specific apoptosis, followed by the strong glial activation, demyelination, and repopulation of OLs. By using this model, we found that CD38 was upregulated in both astrocytes and microglia after CPZ administration. Experiments using wild-type and CD38 knockout (KO) mice, together with those using cultured glial cells, revealed that CD38 deficiency did not affect the initial decrease of the number of OLs, while it attenuated CPZ-induced demyelination, and neurodegeneration. Importantly, the clearance of the degraded myelin and oligodendrocyte repopulation were also reduced in CD38 KO mice. Further experiments revealed that these observations were associated with reduced levels of glial activation and inflammatory responses including phagocytosis, most likely through the enhanced level of NAD+ in CD38-deleted condition. Our results suggest that CD38 and NAD+ in the glial cells play a critical role in the demyelination and subsequent oligodendrocyte remodeling through the modulation of glial activity and neuroinflammation.

Recovery effect of pre-germinated brown rice on the changes of sperm quality, testicular structure and androgen receptor expression in a rat model of drug addiction.

Drug addiction is reported to have adverse effects in male reproduction. Dextromethorphan (DXM) administration was used in this study as a model of addiction in rats, and various treatments including the use of pre-germinated brown rice (PGBR) were investigated for their effects on the changes of sperm quality, testicular structure and androgen receptor (AR) expressions in rats receiving DXM. The results demonstrated that these animals showed significant reduction in all parameters of sperm quality, an increase in abnormal testicular structure and decreased androgen receptor expression in spermatogenic, Sertoli and Leydig cells. However, different effects of the treatments applied in this study were observed with the greatest recovery effect from treatment with PGBR. Sperm motility and sperm concentration reverted to normal after treatment with PGBR for 60 days. Moreover, all parameters of testicular structure also returned to normal after 60 days of PGBR treatment, as well as AR expression in Sertoli and Leydig cells. Therefore, we have demonstrated that PGBR treatment can reverse the changes in sperm quality, testicular structure and AR expression in addicted animals and PGBR may be a novel therapeutic strategy for the treatment of drug addiction.

N-myc downstream-regulated gene 2 protects blood-brain barrier integrity following cerebral ischemia.

Disruption of the blood-brain barrier (BBB) following cerebral ischemia is closely related to the infiltration of peripheral cells into the brain, progression of lesion formation, and clinical exacerbation. However, the mechanism that regulates BBB integrity, especially after permanent ischemia, remains unclear. Here, we present evidence that astrocytic N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, may function as a modulator of BBB permeability following ischemic stroke, using a mouse model of permanent cerebral ischemia. Immunohistological analysis showed that the expression of NDRG2 increases dominantly in astrocytes following permanent middle cerebral artery occlusion (MCAO). Genetic deletion of Ndrg2 exhibited enhanced levels of infarct volume and accumulation of immune cells into the ipsilateral brain hemisphere following ischemia. Extravasation of serum proteins including fibrinogen and immunoglobulin, after MCAO, was enhanced at the ischemic core and perivascular region of the peri-infarct area in the ipsilateral cortex of Ndrg2-deficient mice. Furthermore, the expression of matrix metalloproteinases (MMPs) after MCAO markedly increased in Ndrg2-/- mice. In culture, expression and secretion of MMP-3 was increased in Ndrg2-/- astrocytes, and this increase was reversed by adenovirus-mediated re-expression of NDRG2. These findings suggest that NDRG2, expressed in astrocytes, may play a critical role in the regulation of BBB permeability and immune cell infiltration through the modulation of MMP expression following cerebral ischemia.

Ndrg2 deficiency ameliorates neurodegeneration in experimental autoimmune encephalomyelitis.

N-myc downstream-regulated gene 2 (NDRG2) is a differentiation- and stress-associated molecule that is predominantly expressed in astrocytes in the central nervous system. In this study, we examined the expression and role of NDRG2 in experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. Western blot and immunohistochemical analysis revealed that the expression of NDRG2 was observed in astrocytes of spinal cord, and was enhanced after EAE induction. A comparative analysis of wild-type and Ndrg2-/- mice revealed that deletion of Ndrg2 ameliorated the clinical symptoms of EAE. Although Ndrg2 deficiency only slightly affected the inflammatory response, based on the results of flow cytometry, qRT-PCR, and immunohistochemistry, it significantly reduced demyelination in the chronic phase, and, more importantly, neurodegeneration both in the acute and chronic phases. Further studies revealed that the expression of astrocytic glutamate transporters, including glutamate aspartate transporter (GLAST) and glutamate transporter 1, was more maintained in the Ndrg2-/- mice compared with wild-type mice after EAE induction. Consistent with these results, studies using cultured astrocytes revealed that Ndrg2 gene silencing increased the expression of GLAST, while NDRG2 over-expression decreased it without altering the expression of glial fibrillary acidic protein. The effect of NDRG2 on GLAST expression was associated with the activation of Akt, but not with the activation of nuclear factor-kappa B. These findings suggest that NDRG2 plays a key role in the pathology of EAE by modulating glutamate metabolism. Cover Image for this Issue: doi: 10.1111/jnc.14173.

Accessory hepatic artery arising from celiac trunk: an incidence in a Thai cadaver / Arteria hepática accesoria derivada del tronco celiaco: hallazgo en un cadaver tailandés

An exhaustive knowledge of the liver vascular patterns as well as possible anatomical variations is significant in the planning and performance of all liver surgical procedures in order for the vascularity not to be disturbed or not causing necrosis of the liver parenchyma postoperatively. The celiac trunk usually provides three branches; left gastric, splenic and common hepatic arteries. The left and right hepatic arteries generally derive from proper hepatic artery which is a branch of common hepatic artery. To study the incidence of celiac trunk ramification, the branching patterns of the celiac trunk of 23 Thai cadavers (17 males, 6 females) were documented during routine dissection by medical students at the Department of Anatomy, Faculty of Medical Science, Naresuan University, Thailand. The clinically important variations of the celiac trunk were noted. The results showed that all celiac trunks arose from each aortas at the T12 vertebra (17.39%, 4 cases), intervertebral disc between T12 and L1 vertebra (78.26%, 18 cases) and upper 1/3rd of L1 vertebra (4.35%, 1 case). We found 95.65% (22 cases) normal celiac trunk trifurcation; whereas, 4.35% (1 case) was abnormal quadrifurcation of the trunk. The accessory hepatic artery (aHA) was presented as an additional branch of celiac trunk because the conventional pattern of the left and right hepatic arteries was presented. This finding is one of the rare anatomical variations which is reported in available literatures. The awareness of celiac trunk and its stems aberrant is important in procedures such as liver transplant for appropriate vascular ligation and anastomosis.

Un conocimiento exhaustivo de los patrones vasculares del hígado, así como sus posibles variaciones anatómicas son importantes en la planificación y realización de todos los procedimientos quirúrgicos hepáticos para evitar comprometer la vascularización y posible necrosis del parénquima después de la cirugía. El tronco celíaco, por lo general, proporciona tres ramas: gástrica izquierda, esplénica y arteria hepática común. Las arterias hepáticas izquierda y derecha en general derivan de la arteria hepática propia, que es una rama de la arteria hepática común. El objetivo de este trabajo fue estudiar la incidencia de distribución del tronco celíaco mediante la documentación de patrones de ramificación en 23 cadáveres de Tailandia (17 hombres y 6 mujeres). El estudio se efectuó durante la disección de rutina realizada por los estudiantes de medicina en el Departamento de Anatomía de la Facultad de Ciencias Médicas, Universidad de Naresuan, Tailandia. Se observaron las variaciones clínicamente importantes del tronco celíaco. Los resultados mostraron que todos los troncos celíacos surgieron desde la aorta a nivel de la vértebra T12 (17,39%, 4 casos), a nivel del disco intervertebral entre T12 y L1 vértebra (78,26%, 18 casos) y a nivel del tercio superior de la vértebra L1 (4,35%, 1 caso). Encontramos un 95,65% (22 casos) de troncos celíacos normales, es decir, con trifurcación; mientras que un 4,35% (1 caso) era anormal, con 4 ramos terminales. La arteria hepática accesoria (AHA) se presentó como una rama accesoria del tronco celíaco, ya que existía un patrón convencional de las arterias hepáticas izquierda y derecha. Este hallazgo representa una de las raras variaciones anatómicas informada en la literatura. El conocimiento del tronco celíaco y sus ramas aberrantes son importantes en procedimientos como el trasplante hepático, la anastomosis y una ligadura vascular adecuada.