High Mobility Group A1 Regulates Transcription Levels of Oligodendrocyte Marker Genes in Cultured Oligodendrocyte Precursor Cells.

Oligodendrocyte precursor cells (OPCs) serve as progenitor cells of terminally differentiated oligodendrocytes. Past studies have confirmed the importance of epigenetic system in OPC differentiation to oligodendrocytes. High mobility group A1 (HMGA1) is a small non-histone nuclear protein that binds DNA and modifies the chromatin conformational state. However, it is still completely unknown about the roles of HMGA1 in the process of OPC differentiation. In this study, we prepared primary OPC cultures from the neonatal rat cortex and examined whether the loss- and gain-of-function of HMGA1 would change the mRNA levels of oligodendrocyte markers, such as Cnp, Mbp, Myrf and Plp during the process of OPC differentiation. In our system, the mRNA levels of Cnp, Mbp, Myrf and Plp increased depending on the oligodendrocyte maturation step, but the level of Hmga1 mRNA decreased. When HMGA1 was knocked down by a siRNA approach, the mRNA levels of Cnp, Mbp, Myrf and Plp were smaller in OPCs with Hmga1 siRNA compared to the ones in the control OPCs. On the contrary, when HMGA1 expression was increased by transfection of the Hmga1 plasmid, the mRNA levels of Cnp, Mbp, Myrf and Plp were slightly larger compared to the ones in the control OPCs. These data may suggest that HMGA1 participates in the process of OPC differentiation by regulating the mRNA expression level of myelin-related genes.

Transcriptome Profiling of Mouse Corpus Callosum After Cerebral Hypoperfusion.

White matter damage caused by cerebral hypoperfusion is a major hallmark of subcortical ischemic vascular dementia (SIVD), which is the most common subtype of vascular cognitive impairment and dementia (VCID) syndrome. In an aging society, the number of SIVD patients is expected to increase; however, effective therapies have yet to be developed. To understand the pathological mechanisms, we analyzed the profiles of the cells of the corpus callosum after cerebral hypoperfusion in a preclinical SIVD model. We prepared cerebral hypoperfused mice by subjecting 2-month old male C57BL/6J mice to bilateral carotid artery stenosis (BCAS) operation. BCAS-hypoperfusion mice exhibited cognitive deficits at 4 weeks after cerebral hypoperfusion, assessed by novel object recognition test. RNA samples from the corpus callosum region of sham- or BCAS-operated mice were then processed using RNA sequencing. A gene set enrichment analysis using differentially expressed genes between sham and BCAS-operated mice showed activation of oligodendrogenesis pathways along with angiogenic responses. This database of transcriptomic profiles of BCAS-hypoperfusion mice will be useful for future studies to find a therapeutic target for SIVD.

Mature Adult Mice With Exercise-Preconditioning Show Better Recovery After Intracerebral Hemorrhage.

Background and Purpose: Physical exercise offers therapeutic potentials for several central nervous system disorders, including stroke and cardiovascular diseases. However, it is still mostly unknown whether and how exercise preconditioning affects the prognosis of intracerebral hemorrhage (ICH). In this study, we examined the effects of preconditioning on ICH pathology in mature adult mice using treadmill exercise. Methods: Male C57BL/6J (25-week old) mice were subjected to 6 weeks of treadmill exercise followed by ICH induction. Outcome measurements included various neurological function tests at multiple time points and the assessment of lesion volume at 8 days after ICH induction. In addition, plasma soluble factors and phagocytotic microglial numbers in the peri-lesion area were also measured to determine the mechanisms underlying the effects of exercise preconditioning. Results: The 6-week treadmill exercise preconditioning promoted recovery from ICH-induced neurological deficits in mice. In addition, mice with exercise preconditioning showed smaller lesion volumes and increased numbers of phagocytotic microglia. Furthermore, the levels of several soluble factors, including endostatin, IGFBP (insulin-like growth factor-binding protein)-2 and -3, MMP (matrix metallopeptidase)-9, osteopontin, and pentraxin-3, were increased in the plasma samples from ICH mice with exercise preconditioning compared with ICH mice without exercise. Conclusions: These results suggest that mice with exercise preconditioning may suffer less severe injury from hemorrhagic stroke, and therefore, a habit of physical exercise may improve brain health even in middle adulthood.

Roles of A-kinase Anchor Protein 12 in Astrocyte and Oligodendrocyte Precursor Cell in Postnatal Corpus Callosum.

The formation of the corpus callosum in the postnatal period is crucial for normal neurological function, and clinical genetic studies have identified an association of 6q24-25 microdeletion in this process. However, the mechanisms underlying corpus callosum formation and its critical gene(s) are not fully understood or identified. In this study, we examined the roles of AKAP12 in postnatal corpus callosum formation by focusing on the development of glial cells, because AKAP12 is coded on 6q25.1 and has recently been shown to play roles in the regulations of glial function. In mice, the levels of AKAP12 expression was confirmed to be larger in the corpus callosum compared to the cortex, and AKAP12 levels decreased with age both in the corpus callosum and cortex regions. In addition, astrocytes expressed AKAP12 in the corpus callosum after birth, but oligodendrocyte precursor cells (OPCs), another major type of glial cell in the developing corpus callosum, did not. Furthermore, compared to wild types, Akap12 knockout mice showed smaller numbers of both astrocytes and OPCs, along with slower development of corpus callosum after birth. These findings suggest that AKAP12 signaling may be required for postnatal glial formation in the corpus callosum through cell- and non-cell autonomous mechanisms.

Biphasic roles of pentraxin 3 in cerebrovascular function after white matter stroke.

Recent clinical studies suggest that pentraxin 3 (PTX3), which is known as an acute-phase protein that is produced rapidly at local sites of inflammation, may be a new biomarker of disease risk for central nervous system disorders, including stroke. However, the effects of PTX3 on cerebrovascular function in the neurovascular unit (NVU) after stroke are mostly unknown, and the basic research regarding the roles of PTX3 in NVU function is still limited. In this reverse translational study, we prepared mouse models of white matter stroke by vasoconstrictor (ET-1 or L-Nio) injection into the corpus callosum region to examine the roles of PTX3 in the pathology of cerebral white matter stroke. PTX3 expression was upregulated in GFAP-positive astrocytes around the affected region in white matter for at least 21 days after vasoconstrictor injection. When PTX3 expression was reduced by PTX3 siRNA, blood-brain barrier (BBB) damage at day 3 after white matter stroke was exacerbated. In contrast, when PTX3 siRNA was administered at day 7 after white matter stroke, compensatory angiogenesis at day 21 was promoted. In vitro cell culture experiments confirmed the inhibitory effect of PTX3 in angiogenesis, that is, recombinant PTX3 suppressed the tube formation of cultured endothelial cells in a Matrigel-based in vitro angiogenesis assay. Taken together, our findings may support a novel concept that astrocyte-derived PTX3 plays biphasic roles in cerebrovascular function after white matter stroke; additionally, it may also provide a proof-of-concept that PTX3 could be a therapeutic target for white matter-related diseases, including stroke.

Treadmill Exercise During Cerebral Hypoperfusion Has Only Limited Effects on Cognitive Function in Middle-Aged Subcortical Ischemic Vascular Dementia Mice.

Clinical and basic research suggests that exercise is a safe behavioral intervention and is effective for improving cognitive function in cerebrovascular diseases, including subcortical ischemic vascular dementia (SIVD). However, most of the basic research uses young animals to assess the effects of exercise, although SIVD is an age-related disease. In this study, therefore, we used middle-aged mice to examine how treadmill exercise changes the cognitive function of SIVD mice. As a mouse model of SIVD, prolonged cerebral hypoperfusion was induced in 8-month-old male C57BL/6J mice by bilateral common carotid artery stenosis. A week later, the mice were randomly divided into two groups: a group that received 6-week treadmill exercise and a sedentary group for observation. After subjecting the mice to multiple behavioral tests (Y-maze, novel object recognition, and Morris water maze tests), the treadmill exercise training was shown to only be effective in ameliorating cognitive decline in the Y-maze test. We previously demonstrated that the same regimen of treadmill exercise was effective in young hypoperfused-SIVD mice for all three cognitive tests. Therefore, our study may indicate that treadmill exercise during cerebral hypoperfusion has only limited effects on cognitive function in aging populations.

AKAP12 Supports Blood-Brain Barrier Integrity against Ischemic Stroke.

A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that associates with intracellular molecules to regulate multiple signal transductions. Although the roles of AKAP12 in the central nervous system are still relatively understudied, it was previously shown that AKAP12 regulates blood-retinal barrier formation. In this study, we asked whether AKAP12 also supports the function and integrity of the blood-brain barrier (BBB). In a mouse model of focal ischemia, the expression level of AKAP12 in cerebral endothelial cells was upregulated during the acute phase of stroke. Also, in cultured cerebral endothelial cells, oxygen-glucose deprivation induced the upregulation of AKAP12. When AKAP12 expression was suppressed by an siRNA approach in cultured endothelial cells, endothelial permeability was increased along with the dysregulation of ZO-1/Claudin 5 expression. In addition, the loss of AKAP12 expression caused an upregulation/activation of the Rho kinase pathway, and treatment of Rho kinase inhibitor Y-27632 mitigated the increase of endothelial permeability in AKAP12-deficient endothelial cell cultures. These in vitro findings were confirmed by our in vivo experiments using Akap12 knockout mice. Compared to wild-type mice, Akap12 knockout mice showed a larger extent of BBB damage after stroke. However, the inhibition of rho kinase by Y-27632 tightened the BBB in Akap12 knockout mice. These data may suggest that endogenous AKAP12 works to alleviate the damage and dysfunction of the BBB caused by ischemic stress. Therefore, the AKAP12-rho-kinase signaling pathway represents a novel therapeutic target for stroke.

Different responses after intracerebral hemorrhage between young and early middle-aged mice.

Although aging is a major risk factor for intracerebral hemorrhage (ICH), there are very few studies comparing ICH pathology between young and early middle-aged mice. In this study, 8-month old mice (early middle-aged mice) were compared against 2-month old mice (young mice) in neurological and histological changes after ICH induction, such as body weight, lesion volume, astrocytic responses, and motor and cognitive functions. At day 8 after ICH, there was no significant difference in lesion volume between the two groups, and both groups did not exhibit significant cognitive decline, as assessed by spontaneous alternative Y-maze test. On the other hand, 8-month old mice showed delayed recovery from body weight loss, along with reduced astrocytic activation. Interestingly, in the two motor function tests (beam-walking test and corner turn test), 8-month old mice exhibited lower scores only in the beam-walking test, suggesting a partial disturbance in motor recovery after ICH. These results suggest that age-related differences in ICH pathology may already start to appear in early middle-aged brains.

FGF21 Protects against Aggravated Blood-Brain Barrier Disruption after Ischemic Focal Stroke in Diabetic db/db Male Mice via Cerebrovascular PPARγ Activation.

Recombinant fibroblast growth factor 21 (rFGF21) has been shown to be potently beneficial for improving long-term neurological outcomes in type 2 diabetes mellitus (T2DM) stroke mice. Here, we tested the hypothesis that rFGF21 protects against poststroke blood-brain barrier (BBB) damage in T2DM mice via peroxisome proliferator-activated receptor gamma (PPARγ) activation in cerebral microvascular endothelium. We used the distal middle cerebral occlusion (dMCAO) model in T2DM mice as well as cultured human brain microvascular endothelial cells (HBMECs) subjected to hyperglycemic and inflammatory injury in the current study. We detected a significant reduction in PPARγ DNA-binding activity in the brain tissue and mRNA levels of BBB junctional proteins and PPARγ-targeting gene CD36 and FABP4 in cerebral microvasculature at 24 h after stroke. Ischemic stroke induced a massive BBB leakage two days after stroke in T2DM mice compared to in their lean controls. Importantly, all abnormal changes were significantly prevented by rFGF21 administration initiated at 6 h after stroke. Our in vitro experimental results also demonstrated that rFGF21 protects against hyperglycemia plus interleukin (IL)-1ß-induced transendothelial permeability through upregulation of junction protein expression in an FGFR1 activation and PPARγ activity elevation-dependent manner. Our data suggested that rFGF21 has strong protective effects on acute BBB leakage after diabetic stroke, which is partially mediated by increasing PPARγ DNA-binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

Brief review: Can modulating DNA methylation state help the clinical application of oligodendrocyte precursor cells as a source of stem cell therapy?

Oligodendrocyte precursor cells (OPCs) are one of the major cell types in cerebral white matter, which are generated from neural progenitor cells (NPCs) and give rise to mature oligodendrocytes. Although past studies have extensively examined how OPCs are generated from NPCs and how OPCs differentiate into mature oligodendrocytes, the underlying mechanisms remain unelucidated. In particular, the roles of DNA methylation and the related enzymes DNA methyltransferases (DNMTs) in oligodendrocyte lineage cells are still mostly unknown, although DNA methylation plays a critical role in cell fate decision in multiple cell types. Recently, OPCs were proposed as a promising source of cell-based therapy for patients with oligodendrocyte/myelin damage. Therefore, understanding the mechanisms underlying the involvement of DNMTs in OPCs would help to develop an approach for the efficient preparation of OPCs for cell-based therapy. As a part of the special issue for "Stem Cell Therapy" in Brain Research, this mini-review article first overviews the potential for clinical application of OPCs for cell-based therapy, and then summarizes the key findings of DNMT roles in OPCs, focusing on OPC generation and differentiation.