Global sphingosine-1-phosphate receptor 2 deficiency attenuates neuroinflammation and ischemic-reperfusion injury after neonatal stroke.

Arterial ischemic stroke is common in neonates-1 per 2,300-5,000 births-and therapeutic targets remain insufficiently defined. Sphingosine-1-phosphate receptor 2 (S1PR2), a major regulator of the CNS and immune systems, is injurious in adult stroke. Here, we assessed whether S1PR2 contributes to stroke induced by 3 h transient middle cerebral artery occlusion (tMCAO) in S1PR2 heterozygous (HET), knockout (KO), and wild type (WT) postnatal day 9 pups. HET and WT of both sexes displayed functional deficits in Open Field test whereas injured KO at 24 h reperfusion performed similarly to naives. S1PR2 deficiency protected neurons, attenuated infiltration of inflammatory monocytes, and altered vessel-microglia interactions without reducing increased cytokine levels in injured regions at 72 h. Pharmacologic inhibition of S1PR2 after tMCAO by JTE-013 attenuated injury 72 h after tMCAO. Importantly, the lack of S1PR2 alleviated anxiety and brain atrophy during chronic injury. Altogether, we identify S1PR2 as a potential new target for mitigating neonatal stroke.

Microglia-leucocyte axis in cerebral ischaemia and inflammation in the developing brain.

Development of the Central Nervous System (CNS) is reliant on the proper function of numerous intricately orchestrated mechanisms that mature independently, including constant communication between the CNS and the peripheral immune system. This review summarizes experimental knowledge of how cerebral ischaemia in infants and children alters physiological communication between leucocytes, brain immune cells, microglia and the neurovascular unit (NVU)-the "microglia-leucocyte axis"-and contributes to acute and long-term brain injury. We outline physiological development of CNS barriers in relation to microglial and leucocyte maturation and the plethora of mechanisms by which microglia and peripheral leucocytes communicate during postnatal period, including receptor-mediated and intracellular inflammatory signalling, lipids, soluble factors and extracellular vesicles. We focus on the "microglia-leucocyte axis" in rodent models of most common ischaemic brain diseases in the at-term infants, hypoxic-ischaemic encephalopathy (HIE) and focal arterial stroke and discuss commonalities and distinctions of immune-neurovascular mechanisms in neonatal and childhood stroke compared to stroke in adults. Given that hypoxic and ischaemic brain damage involve Toll-like receptor (TLR) activation, we discuss the modulatory role of viral and bacterial TLR2/3/4-mediated infection in HIE, perinatal and childhood stroke. Furthermore, we provide perspective of the dynamics and contribution of the axis in cerebral ischaemia depending on the CNS maturational stage at the time of insult, and modulation independently and in consort by individual axis components and in a sex dependent ways. Improved understanding on how to modify crosstalk between microglia and leucocytes will aid in developing age-appropriate therapies for infants and children who suffered cerebral ischaemia.

Pharmacological enhancement of retinoid-related orphan receptor α function mitigates spinocerebellar ataxia type 3 pathology.

Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex.

BACKGROUND AND OBJECTIVE: Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice. STUDY DESIGN/MATERIALS AND METHODS: We applied 532 nm LLI (Nd:YVO4, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques. RESULTS: In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons. CONCLUSION: It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.

532 nm low-power laser irradiation recovers γ-secretase inhibitor-mediated cell growth suppression and promotes cell proliferation via Akt signaling.

BACKGROUND AND OBJECTIVE: The γ-secretase inhibitor (GSI) has been shown to inhibit expression of amyloid beta (Aß), but GSI also has a side effect of reducing cell survival. Since low-power laser irradiation (LLI) has been known to promote cell survival, we examined whether 532 nm LLI can rescue the GSI side effect or not. STUDY DESIGN/MATERIALS AND METHODS: The human-derived glioblastoma cells (A-172) were cultured in 35 mm culture dishes or 96-well plate. The center of dish or selected wells was irradiated with 532 nm laser (Nd:YVO4, CW, 60 mW) for 20, 40 and 60 min, respectively. The irradiated cells were photographed at immediately after, 24 and 48 h later and counted. GSI was supplemented in medium 3 h before LLI. The MTT assay was also used to estimate viable cells at 48 h after irradiation. The expression of phosphorylated Akt (p-Akt) or phosphorylated PTEN (p-PTEN) was examined by immunofluorescent staining and measured by fluorescence intensity using the software (BZ-9000, KEYENCE, Japan). RESULTS: GSI application depressed cell proliferation as well as cell survival compared to control. GSI down-regulated Aß but up-regulated p-PTEN and suppressed p-Akt. Application of 532 nm LLI in the presence of GSI significantly recovered the GSI-mediated effects, i.e., LLI could decrease elevated p-PTEN, while increased p-Akt expression with keeping Aß suppression. The LLI effects had a dose-dependency. CONCLUSION: We confirmed that GSI potently suppressed intracellular Aß and decreased cell survival. We conclude that a combination of GSI application and 532 nm LLI can increase cell proliferation via Akt activation while keeping PTEN and Aß suppressed.

Immunocytochemical studies on the effect of 405-nm low-power laser irradiation on human-derived A-172 glioblastoma cells.

The application of low-power laser irradiation (LLI) affects the cell cycle and cell proliferation in various kinds of cells. LLI at a wavelength of 808 nm and a power of 30 mW has been found to significantly decrease the proliferation rate of cells of the human-derived glioblastoma cell line A-172. To determine if this effect of LLI is specific to 808-nm LLI, the present study was designed to reveal the effects of 405-nm LLI under the same experimental conditions. A-172 glioblastoma cells were cultured in 96-well plates according to the conventional protocol. Two different schedules of 405-nm LLI (27 mW) were tested: longer periods of 20, 40 and 60 min and shorter periods of 1, 2, 3, 5, 10 and 15 min. Cells on a digital image displayed on a computer monitor were counted and the proliferation ratio was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) staining. Annexin-V-FLUOS staining and acridine-orange/ethidium-bromide staining were in an immunocytochemical assay to determine if cells were viable or dead (due to apoptosis or necrosis). Cell counting and MTT staining showed that longer 405-nm LLI significantly suppressed the proliferation of A-172 cells at 48 h after LLI (p < 0.05 or p < 0.01) and that the effect of LLI tended to be dose-dependent with morphological changes including cell death. At 90 min after LLI, shorter 405-nm LLI caused necrotic as well as apoptotic cell death, and these effects depended on irradiation time, power and energy density. Detailed analysis revealed that this lethal effect occurred after LLI and was not sustainable. It is concluded that 405-nm LLI has a lethal effect on human-derived glioblastoma A-172 cells, that is different from the suppressive effect without morphological changes induced by 808-nm LLI.