Spatial transcriptomic brain imaging reveals the effects of immunomodulation therapy on specific regional brain cells in a mouse dementia model.

Increasing evidence of brain-immune crosstalk raises expectations for the efficacy of novel immunotherapies in Alzheimer's disease (AD), but the lack of methods to examine brain tissues makes it difficult to evaluate therapeutics. Here, we investigated the changes in spatial transcriptomic signatures and brain cell types using the 10x Genomics Visium platform in immune-modulated AD models after various treatments. To proceed with an analysis suitable for barcode-based spatial transcriptomics, we first organized a workflow for segmentation of neuroanatomical regions, establishment of appropriate gene combinations, and comprehensive review of altered brain cell signatures. Ultimately, we investigated spatial transcriptomic changes following administration of immunomodulators, NK cell supplements and an anti-CD4 antibody, which ameliorated behavior impairment, and designated brain cells and regions showing probable associations with behavior changes. We provided the customized analytic pipeline into an application named STquantool. Thus, we anticipate that our approach can help researchers interpret the real action of drug candidates by simultaneously investigating the dynamics of all transcripts for the development of novel AD therapeutics.

Neovascularization in Outer Membrane of Chronic Subdural Hematoma : A Rationale for Middle Meningeal Artery Embolization.

OBJECTIVE: Chronic subdural hematomas (cSDHs) are generally known to result from traumatic tears of bridging veins. However, the causes of repeat spontaneous cSDHs are still unclear. We investigated the changes in vasculature in the human dura mater and outer membrane (OM) of cSDHs to elucidate the cause of their spontaneous repetition. METHODS: The dura mater was obtained from a normal control participant and a patient with repeat spontaneous cSDHs. The pathological samples from the patient included the dura mater and OM tightly adhered to the inner dura. The samples were analyzed with a particular focus on blood and lymphatic vessels by immunohistochemistry, 3-dimensional imaging using a transparent tissue clearing technique, and electron microscopy. RESULTS: The dural border cell (DBC) layer of the dura mater and OM were histologically indistinguishable. There were 5.9 times more blood vessels per unit volume of tissue in the DBC layer and OM in the patient than in the normal control. The DBC layer and OM contained pathological sinusoidal capillaries not observed in the normal tissue; these capillaries were connected to the middle meningeal arteries via penetrating arteries. In addition, marked lymphangiogenesis in the periosteal and meningeal layers was observed in the patient with cSDHs. CONCLUSION: Neovascularization in the OM seemed to originate from the DBC layer; this is a potential cause of repeat spontaneous cSDHs. Embolization of the meningeal arteries to interrupt the blood supply to pathological capillaries via penetrating arteries may be an effective treatment option.

Spatiotemporal characterization of glial cell activation in an Alzheimer's disease model by spatially resolved transcriptomics.

The molecular changes that occur with the progression of Alzheimer's disease (AD) are well known, but an understanding of the spatiotemporal heterogeneity of changes in the brain is lacking. Here, we investigated the spatially resolved transcriptome in a 5XFAD AD model at different ages to understand regional changes at the molecular level. Spatially resolved transcriptomic data were obtained from 5XFAD AD models and age-matched control mice. Differentially expressed genes were identified using spots clustered by anatomical structures. Gene signatures of activation of microglia and astrocytes were calculated and mapped on the spatially resolved transcriptomic data. We identified early alterations in the white matter (WM) of the AD model before the definite accumulation of amyloid plaques in the gray matter (GM). Changes in the early stage of the disease involved primarily glial cell activation in the WM, whereas the changes in the later stage of pathology were prominent in the GM. We confirmed that disease-associated microglia (DAM) and astrocyte (DAA) signatures also showed initial changes in WM and that activation spreads to GM. Trajectory inference using microglial gene sets revealed the subdivision of DAMs with different spatial patterns. Taken together, these results help to understand the spatiotemporal changes associated with reactive glial cells as a major pathophysiological characteristic of AD. The heterogeneous spatial molecular changes apply to identifying diagnostic and therapeutic targets caused by amyloid accumulation in AD.

Intrathecal [64Cu]Cu-albumin PET reveals age-related decline of lymphatic drainage of cerebrospinal fluid.

Age-related cognitive decline is associated with dysfunctional lymphatic drainage of cerebrospinal fluid (CSF) through meningeal lymphatic vessels. In this study, intrathecal [64Cu]Cu-albumin positron emission tomography (PET) was applied in mice to evaluate lymphatic drainage of CSF and its variation with age. [64Cu]Cu-albumin PET was performed at multiple time points after intrathecal injection of [64Cu]Cu-albumin at an infusion rate of 700 nl/min in adult and aged mice (15-25 months old). CSF clearance and paravertebral lymph nodes were quantified after injection and during the stationary phase. Stationary phase of the next day followed the initial perturbed state by injection of 6 ul (1/7 of total CSF volume) and CSF clearance half-time from the subarachnoid space was 93.4 ± 19.7 and 123.3 ± 15.6 min in adult and aged mice (p = 0.01), respectively. While the % injected dose of CSF space were higher, the activity of the paravertebral lymph nodes were lower in the aged mice on the next day. [64Cu]Cu-albumin PET enabled us to quantify CSF-lymphatic drainage across all levels of brain spinal cords and to visualize and quantify lymph node activity due to CSF drainage. [64Cu]Cu-albumin PET revealed the age-related decrease of the lymphatic drainage of CSF due to this decreased drainage from the subarachnoid space, especially during the stationary phase, in aged mice.

Neuregulin-1 reverses anxiety-like behavior and social behavior deficits induced by unilateral micro-injection of CoCl2 into the ventral hippocampus (vHPC).

Neuregulin-1 (NRG1) is an epidermal growth factor family member with essential roles in the developing and adult nervous systems. In recent years, establishing evidence has collectively suggested that NRG1 is a new modulator of central nervous system (CNS) injury and disease, with multifaceted roles in neuroprotection, remyelination, neuroinflammation, and other repair mechanisms. NRG1 signaling exerts its effects via the tyrosine kinase receptors ErbB2-ErbB4. The NRG1/ErbB network in CNS pathology and repair has evolved, primarily in recent years. In the present study, we demonstrated that a unilateral microinjection of CoCl2 into the ventral hippocampus (vHPC) induced hypoxic insult and led to anxiety-related behaviors and deficit sociability in mice. NRG1 treatment significantly alleviated the CoCl2-induced increase of hypoxic-related molecules and behavioral abnormalities. Furthermore, NRG1 reduced the CoCl2-induced neuroinflammation and neuronal deficits in the vHPC or primary hippocampal neurons in mice. Collectively, these results suggest that NRG1 ameliorates hypoxia by alleviating synaptic deficits and behavioral abnormalities of the CoCl2-induced vHPC hypoxic model.

CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.

BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed. METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF. CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF. Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.

[ 64Cu]Cu-Albumin Clearance Imaging to Evaluate Lymphatic Efflux of Cerebrospinal Space Fluid in Mouse Model.

Purpose: Clearance of brain waste in the cerebrospinal fluid (CSF) through the meningeal lymphatic vessels (mLV) has been evaluated mostly through the fluorescent imaging which has inherent limitations in the context of animal physiology and clinical translatability. The study aimed to establish molecular imaging for the evaluation of mLV clearance function. Methods: Radionuclide imaging after intrathecal (IT) injection was acquired in C57BL/6 mice of 2-9 months. The distribution of [99mTc]Tc-diethylenetriamine pentaacetate (DTPA) and [64Cu]Cu-human serum albumin (HSA) was comparatively evaluated. Evans Blue and [64Cu]Cu-HSA were used to evaluate the distribution of tracer under various speed and volume conditions. Results: [99mTc]Tc-DTPA is not a suitable tracer for evaluation of CSF clearance via mLV as no cervical lymph node uptake was observed while it was cleared from the body. A total volume of 3 to 9 µL at an infusion rate of 300 to 500 nL/min was not sufficient for the tracer to reach the cranial subarachnoid space and clear throughout the mLV. As a result, whole-body positron emission tomography imaging using [64Cu]Cu-HSA at 700 nL/min, to deliver 6 µL of injected volume, was set for characterization of the CSF to mLV clearance. Through this protocol, the mean terminal CSF clearance half-life was measured to be 123.6 min (range 117.0-135.0) in normal mice. Conclusions: We established molecular imaging to evaluate CSF drainage through mLV using [64Cu]Cu-HSA. This imaging method is expected to be extended in animal models of dysfunctional meningeal lymphatic clearance and translational research for disease-modifying therapeutic approaches. Supplementary Information: The online version contains supplementary material available at 10.1007/s13139-022-00746-6.

Human neural stem cell-derived extracellular vesicles protect against Parkinson's disease pathologies.

BACKGROUND: Neural stem cells (NSCs) have the ability to generate a variety of functional neural cell types and have a high potential for neuronal cell regeneration and recovery. Thus, they been recognized as the best source of cell therapy for neurodegenerative diseases, such as Parkinson's disease (PD). Owing to the possibility of paracrine effect-based therapeutic mechanisms and easier clinical accessibility, extracellular vesicles (EVs), which possess very similar bio-functional components from their cellular origin, have emerged as potential alternatives in regenerative medicine. MATERIAL AND METHODS: EVs were isolated from human fibroblast (HFF) and human NSC (F3 cells). The supernatant of the cells was concentrated by a tangential flow filtration (TFF) system. Then, the final EVs were isolated using a total EV isolation kit. RESULTS: In this study, we demonstrate the potential protective effect of human NSC-derived EVs, showing the prevention of PD pathologies in 6-hydroxydopamine (6-OHDA)-induced in vitro and in vivo mouse models. Human NSC and F3 cell (F3)-derived EVs reduced the intracellular reactive oxygen species (ROS) and associated apoptotic pathways. In addition, F3-derived EVs induced downregulation of pro-inflammatory factors and significantly decreased 6-OHDA-induced dopaminergic neuronal loss in vivo. F3 specific microRNAs (miRNAs) such as hsa-mir-182-5p, hsa-mir-183-5p, hsa-mir-9, and hsa-let-7, which are involved in cell differentiation, neurotrophic function, and immune modulation, were found in F3-derived EVs. CONCLUSIONS: We report that human NSC-derived EVs show an effective neuroprotective property in an in vitro transwell system and in a PD model. The EVs clearly decreased ROS and pro-inflammatory cytokines. Taken together, these results indicate that NSC-derived EVs could potentially help prevent the neuropathology and progression of PD.

Extracellular Vesicles Induce an Aggressive Phenotype in Luminal Breast Cancer Cells Via PKM2 Phosphorylation.

BACKGROUND: Aerobic glycolysis is a hallmark of glucose metabolism in cancer. Previous studies have suggested that cancer cell-derived extracellular vesicles (EVs) can modulate glucose metabolism in adjacent cells and promote disease progression. We hypothesized that EVs originating from cancer cells can modulate glucose metabolism in recipient cancer cells to induce cell proliferation and an aggressive cancer phenotype. METHODS: Two breast cancer cell lines with different levels of glycolytic activity, MDA-MB-231 cells of the claudin-low subtype and MCF7 cells of the luminal type, were selected and cocultured as the originating and recipient cells, respectively, using an indirect coculture system, such as a Transwell system or a microfluidic system. The [18F]fluorodeoxyglucose (FDG) uptake by the recipient MCF7 cells was assessed before and after coculture with MDA-MB-231 cells. Proteomic and transcriptomic analyses were performed to investigate the changes in gene expression patterns in the recipient MCF7 cells and MDA-MB-231 cell-derived EVs. RESULTS: FDG uptake by the recipient MCF7 cells significantly increased after coculture with MDA-MB-231 cells. In addition, phosphorylation of PKM2 at tyrosine-105 and serine-37, which is necessary for tumorigenesis and aerobic glycolysis, was highly activated in cocultured MCF7 cells. Proteomic profiling revealed the proliferation and dedifferentiation of MCF7 cells following coculture with MDA-MB-231 cells. Transcriptomic analysis demonstrated an increase in glycolysis in cocultured MCF7 cells, and the component analysis of glycolysis-related genes revealed that the second most abundant component after the cytoplasm was extracellular exosomes. In addition, proteomic analysis of EVs showed that the key proteins capable of phosphorylating PKM2 were present as cargo inside MDA-MB-231 cell-derived EVs. CONCLUSIONS: The phenomena observed in this study suggest that cancer cells can induce a phenotype transition of other subtypes to an aggressive phenotype to consequently activate glucose metabolism via EVs. Therefore, this study could serve as a cornerstone for further research on interactions between cancer cells.

Hippocampal glucose uptake as a surrogate of metabolic change of microglia in Alzheimer's disease.

BACKGROUND: Dynamically altered microglia play an important role in the progression of Alzheimer's disease (AD). Here, we found a close association of the metabolic reconfiguration of microglia with increased hippocampal glucose uptake on [18F]fluorodeoxyglucose (FDG) PET. METHODS: We used an AD animal model, 5xFAD, to analyze hippocampal glucose metabolism using both animal FDG PET and ex vivo FDG uptake test. Cells of the hippocampus were isolated to perform single-cell RNA-sequencing (scRNA-seq). The molecular features of cells associated with glucose metabolism were analyzed at a single-cell level. In order to apply our findings to human brain imaging study, brain FDG PET data obtained from the Alzheimer's Disease Neuroimaging Initiative were analyzed. FDG uptake in the hippocampus was compared according to the diagnosis, AD, mild cognitive impairment, and controls. The correlation analysis between hippocampal FDG uptake and soluble TREM2 in cerebrospinal fluid was performed. RESULTS: In the animal study, 8- and 12-month-old 5xFAD mice showed higher FDG uptake in the hippocampus than wild-type mice. Cellular FDG uptake tests showed that FDG activity in hippocampal microglia was increased in the AD model, while FDG activity in non-microglial cells of the hippocampus was not different between the AD model and wild-type. scRNA-seq data showed that changes in glucose metabolism signatures including glucose transporters, glycolysis and oxidative phosphorylation, mainly occurred in microglia. A subset of microglia with higher glucose transporters with defective glycolysis and oxidative phosphorylation was increased according to disease progression. In the human imaging study, we found a positive association between soluble TREM2 and hippocampal FDG uptake. FDG uptake in the hippocampus at the baseline scan predicted mild cognitive impairment conversion to AD. CONCLUSIONS: We identified the reconfiguration of microglial glucose metabolism in the hippocampus of AD, which could be evaluated by FDG PET as a feasible surrogate imaging biomarker for microglia-mediated inflammation.

Improvement of glymphatic-lymphatic drainage of beta-amyloid by focused ultrasound in Alzheimer's disease model.

Drainage of parenchymal waste through the lymphatic system maintains brain homeostasis. Age-related changes of glymphatic-lymphatic clearance lead to the accumulation beta-amyloid (Aß) in dementia models. In this study, focused ultrasound treatment in combination with microbubbles (FUS-MB) improved Aß drainage in early dementia model mice, 5XFAD. FUS-MB enhanced solute Aß clearance from brain, but not plaques, to cerebrospinal fluid (CSF) space and then deep cervical lymph node (dCLN). dCLN ligation exaggerated memory impairment and progress of plaque formation and also the beneficial effects of FUS-MB upon Aß removal through CSF-lymphatic routes. In this ligation model, FUS-MB improved memory despite accumulation of Aß in CSF. In conclusion, FUS-MB enhances glymphatic-lymphatic clearance of Aß mainly by increasing brain-to-CSF Aß drainage. We suggest that FUS-MB can delay dementia progress in early period and benefits of FUS-MB depend on the effect of Aß disposal through CSF-lymphatics.

Brain Glymphatic/Lymphatic Imaging by MRI and PET.

Since glymphatic was proposed and meningeal lymphatic was discovered, MRI and even PET were introduced to investigate brain parenchymal interstitial fluid (ISF), cerebrospinal fluid (CSF), and lymphatic outflow in rodents and humans. Previous findings by ex vivo fluorescent microscopic, and in vivo two-photon imaging in rodents were reproduced using intrathecal contrast (gadobutrol and the similar)-enhanced MRI in rodents and further in humans. On dynamic MRI of meningeal lymphatics, in contrast to rodents, humans use mainly dorsal meningeal lymphatic pathways of ISF-CSF-lymphatic efflux. In mice, ISF-CSF exchange was examined thoroughly using an intra-cistern injection of fluorescent tracers during sleep, aging, and neurodegeneration yielding many details. CSF to lymphatic efflux is across arachnoid barrier cells over the dorsal dura in rodents and in humans. Meningeal lymphatic efflux to cervical lymph nodes and systemic circulation is also well-delineated especially in humans onintrathecal contrast MRI. Sleep- or anesthesia-related changes of glymphatic-lymphatic flow and the coupling of ISF-CSF-lymphatic drainage are major confounders ininterpreting brain glymphatic/lymphatic outflow in rodents. PET imaging in humans should be interpreted based on human anatomy and physiology, different in some aspects, using MRI recently. Based on the summary in this review, we propose non-invasive and longer-term intrathecal SPECT/PET or MRI studies to unravel the roles of brain glymphatic/lymphatic in diseases.

Maturational delay and asymmetric information flow of brain connectivity in SHR model of ADHD revealed by topological analysis of metabolic networks.

Attention-deficit hyperactivity disorder (ADHD) is a complex brain development disorder characterized by hyperactivity/impulsivity and inattention. A major hypothesis of ADHD is a lag of maturation, which is supported mainly by anatomical studies evaluating cortical thickness. Here, we analyzed changes of topological characteristics of whole-brain metabolic connectivity in twelve SHR rats selected as ADHD-model rats by confirming behavior abnormalities using the marble burying test, open field test, and delay discounting task and 12 Wistar Kyoto rats as the control group, across development from 4 weeks old (childhood) and 6 weeks old (entry of puberty). A topological approach based on graph filtrations revealed a lag in the strengthening of limbic-cortical/subcortical connections in ADHD-model rats. This in turn related to impaired modularization of memory and reward-motivation associated regions. Using mathematical network analysis techniques such as single linkage hierarchical clustering and volume entropy, we observed left-lateralized connectivity in the ADHD-model rats at 6 weeks old. Our findings supported the maturational delay of metabolic connectivity in the SHR model of ADHD, and also suggested the possibility of impaired and compensative reconfiguration of information flow over the brain network.

Graphene oxide-quenching-based fluorescence in situ hybridization (G-FISH) to detect RNA in tissue: Simple and fast tissue RNA diagnostics.

FISH-based RNA detection in paraffin-embedded tissue can be challenging, with complicated procedures producing uncertain results and poor image quality. Here, we developed a robust RNA detection method based on graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) in formalin-fixed paraffin-embedded (FFPE) tissues. Using a fluorophore-labeled peptide nucleic acid (PNA) attached to GO, the endogenous long noncoding RNA BC1, the constitutive protein ß-actin mRNA, and miR-124a and miR-21 could be detected in the cytoplasm of a normal mouse brain, primary cultured hippocampal neurons, an Alzheimer's disease model mouse brain, and glioblastoma multiforme tumor tissues, respectively. Coding and non-coding RNAs, either long or short, could be detected in deparaffinized FFPE or frozen tissues, as well as in clear lipid-exchanged anatomically rigid imaging/immunostaining-compatible tissue hydrogel (CLARITY)-transparent brain tissues. The fluorescence recovered by G-FISH correlated highly with the amount of miR-21, as measured by quantitative real time RT-PCR. We propose G-FISH as a simple, fast, inexpensive, and sensitive method for RNA detection, with a very low background, which could be applied to a variety of research or diagnostic purposes.

Development of tau PET Imaging Ligands and their Utility in Preclinical and Clinical Studies.

The pathological features of Alzheimer's disease are senile plaques which are aggregates of ß-amyloid peptides and neurofibrillary tangles in the brain. Neurofibrillary tangles are aggregates of hyperphosphorylated tau proteins, and these induce various other neurodegenerative diseases, such as progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and chronic traumatic encephalopathy. In the case of Alzheimer's disease, the measurement of neurofibrillary tangles associated with cognitive decline is suitable for differential diagnosis, disease progression assessment, and to monitor the effects of therapeutic treatment. This review discusses considerations for the development of tau ligands for imaging and summarizes the results of the first-in-human and preclinical studies of the tau tracers that have been developed thus far. The development of tau ligands for imaging studies will be helpful for differential diagnosis and for the development of therapeutic treatments for tauopathies including Alzheimer's disease.

Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs.

Fluorescence imaging of in vivo miR-124a-induced neurogenesis of neuronal progenitor cells using neuron-specific reporters.

BACKGROUND: Facilitation of the differentiation of the stem cells toward neuronal lineage is crucial for enhancing the differentiation efficacy of grafted stem cells for the possible treatment of neurodegenerative disorders. MicroRNA124a (miR-124a) has been considered as a neuronal lineage regulator, possessing the capability to activate neuronal differentiation. In this study, using a neuronal promoter-based reporter and live-cell fluorescence imaging, we visualized in vitro and in vivo the enhanced neuronal differentiation of neuronal progenitor cells with miR-124a overproduction. METHODS: The neuron specific alpha1 tubulin promoter-driven RFP reporter (pTa1-RFP) was used to trace the miR-124a-induced neuronal differentiation in live cell condition. MiR-124a or miR-scramble in 10 % glucose buffer was mixed with in vivo-jetPEITM and in vivo fluorescence images were obtained daily using Maestro spectral fluorescent imager. RESULTS: Neurite outgrowth was clearly seen in F11 cells after miR-124a transfection, and immunofluorescence staining showed increase of Tuj1 and NF at 48 hours. When pTa1-RFP-transfected F11 cells were implanted simultaneously with miR-124a into the nude mice, gradually increasing reporter signals and morphological changes indicated neuronal differentiation for 48 hours in live cells in vitro. The miR-124a-treated F11 cells showed higher reporter signals on in vivo fluorescence imaging than miR-scramble-treated cells, which were verified by ex vivo confirmation of Tuj1 and NF expression. CONCLUSIONS: These results indicated that neuronal reporter-based neurogenesis imaging can be used for monitoring miR-124a acting as neuronal activator when miRNA was injected in in vivo PEI-coated form for miRNA-mediated regenerative therapy.

Detection of intra-brain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector.

Detection of cellular expression of long noncoding RNAs (lncRNAs) was elusive due to the ambiguity of exposure of their reactive sequences associated with their secondary/tertiary structures and dynamic binding of proteins around lncRNAs. Herein, we developed graphene-based detection techniques exploiting the quenching capability of graphene oxide (GO) flakes for fluorescent dye (FAM)-labeled single-stranded siRNAs and consequent un-quenching by their detachment from GO by matching lncRNAs. A brain cytoplasmic 1 (BC1) lncRNA expression was significantly decreased by a siRNA, siBC1-1. GO quenched the FAM-labeled siBC1-1 peptide nucleic acid (PNA) probe, and this quenching was recovered by BC1. While FAM-siBC1-1-PNA-GO complex transfected spontaneously mouse or human neural stem cells, fluorescence was recovered only in mouse cells having high BC1 expression. Fluorescent dye-labeled single-stranded RNA-GO probe could detect the reactive exposed nucleic acid sequence of a cytoplasmic lncRNA expressing in the cytoplasm, which strategy can be used as a detection method of lncRNA expression.

Maturation of metabolic connectivity of the adolescent rat brain.

Neuroimaging has been used to examine developmental changes of the brain. While PET studies revealed maturation-related changes, maturation of metabolic connectivity of the brain is not yet understood. Here, we show that rat brain metabolism is reconfigured to achieve long-distance connections with higher energy efficiency during maturation. Metabolism increased in anterior cerebrum and decreased in thalamus and cerebellum during maturation. When functional covariance patterns of PET images were examined, metabolic networks including default mode network (DMN) were extracted. Connectivity increased between the anterior and posterior parts of DMN and sensory-motor cortices during maturation. Energy efficiency, a ratio of connectivity strength to metabolism of a region, increased in medial prefrontal and retrosplenial cortices. Our data revealed that metabolic networks mature to increase metabolic connections and establish its efficiency between large-scale spatial components from childhood to early adulthood. Neurodevelopmental diseases might be understood by abnormal reconfiguration of metabolic connectivity and efficiency.

Neuritin attenuates cognitive function impairments in tg2576 mouse model of Alzheimer's disease.

Neuritin, also known as CPG15, is a neurotrophic factor that was initially discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in the process of neural development and synaptic plasticity, although its binding receptor(s) and downstream signaling effectors remain unclear. In this study, we found that the cortical and hippocampal expression of neuritin is reduced in the brains of Alzheimer's disease (AD) patients and demonstrated that viral-mediated expression of neuritin in the dentate gyrus of 13-month-old Tg2576 mice, an AD animal model, attenuated a deficit in learning and memory as assessed by a Morris water maze test. We also found that neuritin restored the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neuron cultures prepared from Tg2576 mice. It was also shown that viral-mediated expression of neuritin in the dentate gyrus of 7-week-old Sprague-Dawley rats increased neurogenesis in the hippocampus. Taken together, our results demonstrate that neuritin restores the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neurons from Tg2576 neurons, and also attenuates cognitive function deficits in Tg2576 mouse model of AD, suggesting that neuritin possesses a therapeutic potential for AD.