A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction.

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell-cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell-cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell-cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.

Gap junction-mediated cell-cell interaction between transplanted mesenchymal stem cells and vascular endothelium in stroke.

We have shown previously that transplanted bone marrow mononuclear cells (BM-MNC), which are a cell fraction rich in hematopoietic stem cells, can activate cerebral endothelial cells via gap junction-mediated cell-cell interaction. In the present study, we investigated such cell-cell interaction between mesenchymal stem cells (MSC) and cerebral endothelial cells. In contrast to BM-MNC, for MSC we observed suppression of vascular endothelial growth factor uptake into endothelial cells and transfer of glucose from endothelial cells to MSC in vitro. The transfer of such a small molecule from MSC to vascular endothelium was subsequently confirmed in vivo and was followed by suppressed activation of macrophage/microglia in stroke mice. The suppressive effect was absent by blockade of gap junction at MSC. Furthermore, gap junction-mediated cell-cell interaction was observed between circulating white blood cells and MSC. Our findings indicate that gap junction-mediated cell-cell interaction is one of the major pathways for MSC-mediated suppression of inflammation in the brain following stroke and provides a novel strategy to maintain the blood-brain barrier in injured brain. Furthermore, our current results have the potential to provide a novel insight for other ongoing clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, as well as other diseases such as COVID-19 (coronavirus disease 2019).

Liver-specific dysregulation of clock-controlled output signal impairs energy metabolism in liver and muscle.

The liver is the major organ maintaining metabolic homeostasis in animals during shifts between fed and fasted states. Circadian oscillations in peripheral tissues including the liver are connected with feeding-fasting cycles. We generated transgenic mice with hepatocyte specific E4BP4, D-box negative regulator, overexpression. Liver-specific E4BP4 overexpression was also achieved by adenoviral gene transfer. Interestingly, hepatic E4BP4 overexpression induced marked insulin resistance, that was rescued by DBP, a competing D-box positive regulator, overexpression. At basal conditions hepatocyte E4BP4 transgenic mice exhibited increased gluconeogenesis with reduced AKT phosphorylation in liver. In muscle, AKT phosphorylation was impaired after insulin stimulation. Such muscle insulin resistance was associated with elevated free fatty acid flux from the liver and reduced fatty acid utilization as an energy source during the inactive phase. E4BP4, one of the clock-controlled output genes, are key metabolic regulators in liver adjusting liver and muscle metabolism and insulin sensitivity in the feeding-fasting cycles. Its tuning is critical for preventing metabolic disorders.

Bone Marrow Mononuclear Cells Transplantation and Training Increased Transplantation of Energy Source Transporters in Chronic Stroke.

OBJECTIVES: Bone marrow mononuclear cells (BM-MNC) show a significant therapeutic effect in combination with training even in the chronic phase of stroke. However, the mechanism of this combination therapy has not been investigated. Here, we examined its effects on brain metabolism in chronic stroke mice. MATERIALS AND METHODS: BM-MNC (1x105 cells in 100 µL of phosphate-buffered saline) were intravenously transplanted at 4 weeks (chronic stage) after the middle cerebral artery occlusion. At 3 h and 10 weeks after the administration of BM-MNC, we evaluated transcription changes of the metabolism-related genes, hypoxia inducible factor 1-α (Hif-1α), prolyl hydroxylase 3 (Phd3), pyruvate dehydrogenase kinase 1 (Pdk1), Na+/K+-ATPase (Atp1α1‒3), connexins, glucose transporters, and monocarboxylate transporters, in the brain during chronic phase of stroke using quantitative polymerase chain reaction. RESULTS: The results showed transcriptional activation of the metabolism-related genes in the contralateral cortex at 3 h after BM-MNC transplantation. Behavioral tests were performed after cell therapy, and the brain metabolism of mice with improved motor function was examined at 10 weeks after cell therapy. The therapeutic efficacy of the combination therapy with BM-MNC transplantation and training was evident in the form of transcriptional activation of ipsilateral anterior cerebral artery (ACA) cortex. CONCLUSIONS: BM-MNC transplantation combined with training for chronic stroke activated gene expression in both the ipsilateral and the contralateral side.

Bone Marrow Mononuclear Cells Activate Angiogenesis via Gap Junction-Mediated Cell-Cell Interaction.

Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.

Clot-Derived Contaminants in Transplanted Bone Marrow Mononuclear Cells Impair the Therapeutic Effect in Stroke.

Background and Purpose- The beneficial effects of bone marrow mononuclear cell (BM-MNC) transplantation in preclinical experimental stroke have been reliably demonstrated. However, only overall modest effects in clinical trials were observed. We have investigated and reported a cause of the discrepancy between the preclinical and clinical studies. Methods- To investigate the possible cause of low efficacy of BM-MNC transplantation in experimental stroke, we have focused on blood clot formation, which is not uncommon in human bone marrow aspirates. To evaluate the effects of clot-derived contaminants in transplanted BM-MNC on stroke outcome, a murine stroke model was used. Results- We show that BM-MNC separated by an automatic cell isolator (Sepax2), which does not have the ability to remove clots, did not attenuate brain atrophy after stroke. In contrast, manually isolated, clot-free BM-MNC exerted therapeutic effects. Clot-derived contaminants were also transplanted intravenously to poststroke mice. We found that the transplanted contaminants were trapped at the peristroke area, which were associated with microglial/macrophage activation. Conclusions- Clot-derived contaminants in transplanted BM-MNC nullify therapeutic effects in experimental stroke. This may explain neutral results in clinical trials, especially in those using automated stem cell separators that lack the ability to remove clot-derived contaminants. Visual Overview- An online visual overview is available for this article.

Amyloid ß deposition in subcortical stroke patients and effects of educational achievement: A pilot study.

OBJECTIVE: A direct causal relationship of cerebrovascular risk factors/stroke to amyloid ß (Aß) deposition has yet to be shown. We conducted [11 C] Pittsburgh compound B (PiB)-positron emission tomography (PET) analysis on subacute ischemic stroke patients and healthy controls. We hypothesized that subacute ischemic stroke patients would show focal Aß accumulation in cortical regions, which would increase and extend over time during the chronic phase after stroke onset. METHODS: Patients were recruited 14 to 28 days after acute subcortical ischemic stroke and examined with [11 C]PiB-PET scans. Regional time-activity data were analyzed with the Logan graphical method. Whole brain voxel-based analysis was conducted to compare stroke patients with healthy controls. We also performed longitudinal comparison of patients with successive [11 C]PiB-PET scans 1 year after stroke. RESULTS: Voxel-based analysis revealed a significant increase of [11 C]PiB-BPND of the precuneus/posterior cingulate cortex (PCu/PCC) in stroke patients at the subacute stage. Based on stepwise multiple regression analysis of [11 C]PiB-BP changes during follow-up as the dependent variable, years of education was the best independent correlate. There was a significant negative relationship between changes in [11 C]PiB-BP and years of education. CONCLUSIONS: Our results suggest that processes before and after the onset of ischemic stroke may trigger Aß deposition in the PCu/PCC, whereby amyloid deposition begins at an early stage of Alzheimer's disease (AD). Our findings support the existence of a cooperative association between vascular risk factors/stroke and AD progression. Further, educational achievement had a protective effect against the increase in Aß accumulation.

Mutual effect of cerebral amyloid ß and peripheral lymphocytes in cognitively normal older individuals.

OBJECTIVE: We hypothesized that cerebral amyloid accumulation is reflected in the periphery in the pre-dementia stage and used flow cytometry to investigate the peripheral lymphocytes as an easily accessible biomarker to observe neuro-inflammation. We aimed to determine whether peripheral lymphocytes are related to the cortical amyloid burden or vice versa in cognitively normal older subjects. METHODS: We applied [11 C] Pittsburgh compound B (PiB)-positron emission tomography to 36 cognitively normal older individuals, and Aß deposition was quantified by cortical binding potential (PiB-BPND ). Blood samples were obtained, and lymphocyte subsets were evaluated. We examined differences between low and high PiB-BPND groups in the percentage of B cells, T cells, helper T cells, cytotoxic T cells, regulatory T cells, and natural killer cells. RESULTS: Subjects with high PiB-BPND showed significantly higher percentage of cytotoxic T cells (%CD3+ ). Correlation analysis revealed a significant relationship between the percentage of cytotoxic T cells and global cortical mean PiB-BPND . Hierarchical regression analyses showed that cytotoxic T cells were significantly related to the value of global cortical mean PiB-BPND and vice versa. CONCLUSIONS: Our results indicated that a specific peripheral immune response, reflected in the increased ratio of cytotoxic T cells, could be regarded as a preclinical sign of AD and could be attributed to the Aß neuropathological mechanism. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluations of Intravenous Administration of CD34+ Human Umbilical Cord Blood Cells in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34+ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term "neonatal encephalopathy," their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

High amyloid-ß deposition related to depressive symptoms in older individuals with normal cognition: a pilot study.

OBJECTIVE: Previous studies have reported depressive symptoms in the preclinical stages of Alzheimer's disease (AD). The objective of this study was to determine whether depressive symptoms are associated with cortical amyloid burden. In order to do this, we measured cortical amyloid via (11) C-labeled Pittsburgh Compound B ([(11) C]PIB) uptake using positron emission tomography (PET) in cognitively normal subjects. METHODS: We performed [(11) C]PIB-PET in 29 cognitively normal, older participants. Depressive symptoms were assessed using the 15-item Geriatric Depression Scale (GDS). Aß deposition was quantified by binding potential (BPND ), and the association between cortical mean BPND values and GDS scores was evaluated. Analysis of parametric BPND images was performed to examine the relationship between regional BPND and GDS scores. RESULTS: We found a positive correlation between depressive symptoms and mean cortical PIB-BPND in groups of subjects with middle to high PIB-BPND . There was little change in GDS-depression score between subjects with low and middle PIB-BPND levels, while an increase in GDS was shown in the high PIB-BPND group. The main BPND increase was localized to the precuneus/posterior cingulate cortex (PCu/PCC) in subjects with high PIB-BPND , and we found a significant positive relationship between PIB-BPND in this area and depressive symptoms. CONCLUSIONS: Emotional dysregulation because of Aß neuropathology in the PCu/PCC may relate to depressive symptoms. More specifically, we found that older, cognitively normal patients with depressive episodes were more likely to have underlying AD pathology. Thus, depressive symptoms may increase the predictive ability of the identification of future AD cases. Copyright © 2016 John Wiley & Sons, Ltd.

Low amyloid-ß deposition correlates with high education in cognitively normal older adults: a pilot study.

OBJECTIVE: Several epidemiological studies have found a lower incidence of Alzheimer's disease in highly educated populations, but the protective mechanism of education against the disease is still unclear. Our objective was to investigate the association between education and (11) C-labeled Pittsburgh Compound B (PIB) uptake with positron emission tomography in participants with normal cognitive ability. METHODS: We performed (11) C-labeled PIB positron emission tomography and neuropsychological testing in 30 cognitively normal older participants. Of the participants, 16 had a period of education less than 12 years (low-education group) and 14 had more than 13 years (high-education group). Amyloid-ß deposition was quantified by binding potential (BPND ) in several brain regions and was compared between the groups with different education levels. RESULTS: We found significantly higher cortical PIB-BPND in the cognitively normal participants with low education compared with the ones with high education. None of the brain regions in low-education group showed significantly lower BPND values. This finding was not affected by the inclusion of possible confounding variables such as age, sex, and general intelligence. Our findings indicated a reduced amyloid pathology in highly educated, cognitively normal, participants. CONCLUSIONS: Our findings lead to the proposal that early-life education has a negative association with Alzheimer's disease pathology. This proposal is not in opposition to the brain reserve hypothesis. People with more education might be prone to a greater inhibitory effect against amyloid-ß deposition before the preclinical stage. At the same time, they have a greater reserve capacity, and greater pathological changes are required for dementia to manifest.

Delayed atrophy in posterior cingulate cortex and apathy after stroke.

OBJECTIVE: A few studies have been performed on chronic structural changes after stroke. The primary purpose of the present study was to investigate regional cortical volume changes after the onset of stroke and to examine how the cortical volume changes affected neuropsychiatric symptoms. METHODS: Participants were 20 stroke patients and 14 control subjects. T1-MRI was performed twice, once at the subacute stage and again 6 months later, and whole brain voxel-based morphometric (VBM) analysis was used to detect significant cortical gray matter volume changes in patients. We also assessed the correlation between changes in cortical volumes and changes in neuropsychiatric symptoms during the 6 months following a stroke. RESULTS: In the present study, we found significant volume reductions in the anterior part of the posterior cingulate cortex (PCC) over the 6 months following a stroke by exploratory VBM analysis. We also found that the amount of volume change was significantly correlated with the change in apathy-scale scores during the 6 months poststroke. CONCLUSIONS: The present study suggests that delayed atrophic change is evident in the PCC 6 months after a stroke. There was greater apathetic change in the stroke patients with the larger volume reductions. The delayed atrophy of the PCC may reflect degeneration secondary to neuronal loss due to stroke. Such degeneration might have impaired control of goal-directed behavior, leading to the observed increase in apathy.

Microstructural abnormality in white matter, regulatory T lymphocytes, and depressive symptoms after stroke.

BACKGROUND: The purpose of the present study was to investigate the existence of microstructure abnormalities in the white matter circuit in stroke patients and its relationship to depressive episodes. To target the prevention of depression, we also investigated the relationship between lymphocyte subsets and cerebral abnormalities in patients. METHODS: Participants included 18 patients with acute ischemic stroke and 22 healthy control subjects. Diffusion tensor imaging was performed. Whole-brain voxel-based analysis was used to compare fractional anisotropy (FA) between groups. Blood samples were obtained, and the lymphocyte subsets were evaluated using flow cytometry. Follow-up examinations were conducted on 12 patients at 6 months. RESULTS: FA was decreased in the bilateral anterior limb of the internal capsule in stroke patients. At the 6-month follow-up examination, there was a significant increase in FA, which was associated with a lower depression scale score. Patients showed a decreased percentage of circulated regulatory T lymphocytes, and the degree of reduction was related to the decrease in the FA value in the internal capsule. CONCLUSIONS: FA reductions in the anterior limb of the internal capsule cause abnormality in the frontal-subcortical circuits and confer a biological vulnerability, which in combination with environmental stressors results in the onset of depression. Our findings also demonstrated the possibility of preventing post-stroke depression by targeting the role of regulatory T lymphocytes in brain tissue repair and regeneration after stroke.

RNA Analysis of Circulating Leukocytes in Patients with Alzheimer's Disease.

Background: One of the key symptoms of Alzheimer's disease (AD) is the impairment of short-term memory. Hippocampal neurogenesis is essential for short-term memory and is known to decrease in patients with AD. Impaired short-term memory and impaired neurogenesis are observed in aged mice alongside changes in RNA expression of gap junction and metabolism-related genes in circulating leukocytes. Moreover, after penetrating the blood-brain barrier via the SDF1/CXCR4 axis, circulating leukocytes directly interact with hippocampal neuronal stem cells via gap junctions. Objective: Evaluation of RNA expression profiles in circulating leukocytes in patients with AD. Methods: Patients with AD (MMSE≧23, n = 10) and age-matched controls (MMSE≧28, n = 10) were enrolled into this study. RNA expression profiles of gap junction and metabolism-related genes in circulating leukocytes were compared between the groups (jRCT: 1050210166). Results: The ratios of gap junction and metabolism-related genes were significantly different between patients with AD and age-matched controls. However, due to large inter-individual variations, there were no statistically significant differences in the level of single RNA expression between these groups. Conclusions: Our findings suggest a potential connection between the presence of circulating leukocytes and the process of hippocampal neurogenesis in individuals with AD. Analyzing RNA in circulating leukocytes holds promise as a means to offer novel insights into the pathology of AD, distinct from conventional markers.