Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE) 1-8 . However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY + p300 + memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY + p300 + astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro ; scRNA-seq and immunohistochemistry studies detected increased ACLY + p300 + astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

Neuroinflammation: An astrocyte perspective.

Astrocytes are abundant glial cells in the central nervous system (CNS) that play active roles in health and disease. Recent technologies have uncovered the functional heterogeneity of astrocytes and their extensive interactions with other cell types in the CNS. In this Review, we highlight the intricate interactions between astrocytes, other CNS-resident cells, and CNS-infiltrating cells as well as their potential therapeutic value in the context of inflammation and neurodegeneration.

Cerebellar granule cell signaling is indispensable for normal motor performance.

Within the cerebellar cortex, mossy fibers (MFs) excite granule cells (GCs) that excite Purkinje cells (PCs), which provide outputs to the deep cerebellar nuclei (DCNs). It is well established that PC disruption produces motor deficits such as ataxia. This could arise from either decreases in ongoing PC-DCN inhibition, increases in the variability of PC firing, or disruption of the flow of MF-evoked signals. Remarkably, it is not known whether GCs are essential for normal motor function. Here we address this issue by selectively eliminating calcium channels that mediate transmission (CaV2.1, CaV2.2, and CaV2.3) in a combinatorial manner. We observe profound motor deficits but only when all CaV2 channels are eliminated. In these mice, the baseline rate and variability of PC firing are unaltered, and locomotion-dependent increases in PC firing are eliminated. We conclude that GCs are indispensable for normal motor performance and that disruption of MF-induced signals impairs motor performance.

Adsorption Phenomenon of VOCs Released from the Fiber-Reinforced Plastic Production onto Carbonaceous Surface.

The manufacturing of fiber-reinforced plastics has been linked to the discharge of volatile organic compounds (VOCs), particularly toluene and benzene, which have been identified as posing substantial risks to human health and the environment. To counteract this issue, activated carbons have been suggested as a means of reducing VOC emissions through adsorption. The objective of this study was to investigate the adsorption characteristics of toluene and benzene onto activated carbons produced from coal (AC) and coconut shells (CAC). The study was carried out in an aqueous medium. The findings revealed that the AC sample with higher surface characteristics exhibited a higher adsorption capacity (toluene: 196.0784 mg g-1 and benzene: 181.8182 mg g-1) in comparison to the CAC sample (toluene: 135.1351 mg g-1 and benzene: 116.2791 mg g-1). The superior adsorption performance of AC on both VOCs can be attributed to its higher surface characteristics. The Langmuir model was found to be more appropriate than the Freundlich model, as indicated by the higher coefficient of determination (R2) value of the Langmuir isotherm (avg. R2 = 0.9669) compared to that of the Freundlich isotherm (avg. R2 = 0.9654), suggesting the use of a monolayer adsorption mechanism. The adsorption kinetics of the samples were analyzed using the pseudo-first-order and pseudo-second-order models, and the former was found to be more fitting, indicating that the rate of adsorption is directly proportional to the concentration difference between the solution and the sample surface. The adsorption process was found to be spontaneous and favorable based on the positive value of ΔG_ads. Furthermore, the adsorption process was endothermic and disordered, as indicated by the positive values of ΔH_ads and ΔS_ads. The regeneration efficiency of all the samples was secured more than 95% upon the fifth cycle.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Bio-Degradable Polyesters with Rigid Cyclic Diester from Camphor and Tartaric Acid.

Despite their excellent, useful, and stable properties, thermoplastics are constantly subject to environmental risks because of their low degradability under thermal, chemical, and mechanical stresses. To overcome the aforementioned issues, we hereby introduce an eco-friendly camphor (Ct) cyclic diester. The Ct diester is designed as a monomer, including a ketal group from the Ct, and shows high thermal stability via a rigid spiro-ring and a bridged bicyclic structure. A series of polyester was synthesized using the Ct diester, including various types of diols and dimethyl terephthalate. PETxCty copolyesters showed appropriate thermal stability up to 414 °C and a high glass transition temperature. This thermal behavior led to amorphous regions as the Ct diester content increased. Regarding the proportion of the Ct diester in the polyester, it was sensitive to hydrolysis and contributed to the degradation of the polyester in acid buffer conditions.

Stroke subtype-dependent synapse elimination by reactive gliosis in mice.

The pathological role of reactive gliosis in CNS repair remains controversial. In this study, using murine ischemic and hemorrhagic stroke models, we demonstrated that microglia/macrophages and astrocytes are differentially involved in engulfing synapses in the reactive gliosis region. By specifically deleting MEGF10 and MERTK phagocytic receptors, we determined that inhibiting phagocytosis of microglia/macrophages or astrocytes in ischemic stroke improved neurobehavioral outcomes and attenuated brain damage. In hemorrhagic stroke, inhibiting phagocytosis of microglia/macrophages but not astrocytes improved neurobehavioral outcomes. Single-cell RNA sequencing revealed that phagocytosis related biological processes and pathways were downregulated in astrocytes of the hemorrhagic brain compared to the ischemic brain. Together, these findings suggest that reactive microgliosis and astrogliosis play individual roles in mediating synapse engulfment in pathologically distinct murine stroke models and preventing this process could rescue synapse loss.

Excitatory neuronal CHD8 in the regulation of neocortical development and sensory-motor behaviors.

CHD8 (chromodomain helicase DNA-binding protein 8) is a chromatin remodeler associated with autism spectrum disorders. Homozygous Chd8 deletion in mice leads to embryonic lethality, making it difficult to assess whether CHD8 regulates brain development and whether CHD8 haploinsufficiency-related macrocephaly reflects normal CHD8 functions. Here, we report that homozygous conditional knockout of Chd8 restricted to neocortical glutamatergic neurons causes apoptosis-dependent near-complete elimination of neocortical structures. These mice, however, display normal survival and hyperactivity, anxiolytic-like behavior, and increased social interaction. They also show largely normal auditory function and moderately impaired visual and motor functions but enhanced whisker-related somatosensory function. These changes accompany thalamic hyperactivity, revealed by 15.2-Tesla fMRI, and increased intrinsic excitability and decreased inhibitory synaptic transmission in thalamic ventral posterior medial (VPM) neurons involved in somatosensation. These results suggest that excitatory neuronal CHD8 critically regulates neocortical development through anti-apoptotic mechanisms, neocortical elimination distinctly affects cognitive behaviors and sensory-motor functions in mice, and Chd8 haploinsufficiency-related macrocephaly might represent compensatory responses.

Astrocytes phagocytose adult hippocampal synapses for circuit homeostasis.

In the adult hippocampus, synapses are constantly formed and eliminated1,2. However, the exact function of synapse elimination in the adult brain, and how it is regulated, are largely unknown. Here we show that astrocytic phagocytosis3 is important for maintaining proper hippocampal synaptic connectivity and plasticity. By using fluorescent phagocytosis reporters, we find that excitatory and inhibitory synapses are eliminated by glial phagocytosis in the CA1 region of the adult mouse hippocampus. Unexpectedly, we found that astrocytes have a major role in the neuronal activity-dependent elimination of excitatory synapses. Furthermore, mice in which astrocytes lack the phagocytic receptor MEGF10 show a reduction in the elimination of excitatory synapses; as a result, excessive but functionally impaired synapses accumulate. Finally, Megf10-knockout mice show defective long-term synaptic plasticity and impaired formation of hippocampal memories. Together, our data provide strong evidence that astrocytes eliminate unnecessary excitatory synaptic connections in the adult hippocampus through MEGF10, and that this astrocytic function is crucial for maintaining circuit connectivity and thereby supporting cognitive function.

The rostroventral part of the thalamic reticular nucleus modulates fear extinction.

The thalamus has been implicated in fear extinction, yet the role of the thalamic reticular nucleus (TRN) in this process remains unclear. Here, in mice, we show that the rostroventral part of the TRN (TRNrv) is critically involved in the extinction of tone-dependent fear memory. Optogenetic excitation of TRNrv neurons during extinction learning dramatically facilitated, whereas the inhibition disrupted, the fear extinction. Single unit recordings demonstrated that TRNrv neurons selectively respond to conditioned stimuli but not to neutral stimuli. TRNrv neurons suppressed the spiking activity of the medial part of the dorsal midline thalamus (dMTm), and a blockade of this inhibitory pathway disrupted fear extinction. Finally, we found that the suppression of dMTm projections to the central amygdala promotes fear extinction, and TRNrv neurons have direct connections to this pathway. Our results uncover a previously unknown function of the TRN and delineate the neural circuit for thalamic control of fear memory.

Human glioblastoma arises from subventricular zone cells with low-level driver mutations.

Glioblastoma (GBM) is a devastating and incurable brain tumour, with a median overall survival of fifteen months1,2. Identifying the cell of origin that harbours mutations that drive GBM could provide a fundamental basis for understanding disease progression and developing new treatments. Given that the accumulation of somatic mutations has been implicated in gliomagenesis, studies have suggested that neural stem cells (NSCs), with their self-renewal and proliferative capacities, in the subventricular zone (SVZ) of the adult human brain may be the cells from which GBM originates3-5. However, there is a lack of direct genetic evidence from human patients with GBM4,6-10. Here we describe direct molecular genetic evidence from patient brain tissue and genome-edited mouse models that show astrocyte-like NSCs in the SVZ to be the cell of origin that contains the driver mutations of human GBM. First, we performed deep sequencing of triple-matched tissues, consisting of (i) normal SVZ tissue away from the tumour mass, (ii) tumour tissue, and (iii) normal cortical tissue (or blood), from 28 patients with isocitrate dehydrogenase (IDH) wild-type GBM or other types of brain tumour. We found that normal SVZ tissue away from the tumour in 56.3% of patients with wild-type IDH GBM contained low-level GBM driver mutations (down to approximately 1% of the mutational burden) that were observed at high levels in their matching tumours. Moreover, by single-cell sequencing and laser microdissection analysis of patient brain tissue and genome editing of a mouse model, we found that astrocyte-like NSCs that carry driver mutations migrate from the SVZ and lead to the development of high-grade malignant gliomas in distant brain regions. Together, our results show that NSCs in human SVZ tissue are the cells of origin that contain the driver mutations of GBM.

Stereotactic ablative body radiotherapy for spinal metastasis from hepatocellular carcinoma: its oncologic outcomes and risk of vertebral compression fracture.

Spinal metastases from hepatocellular carcinoma (HCC) require high-dose irradiation for durable pain and tumor control. Stereotactic ablative body radiotherapy (SABR) enables the delivery of high-dose radiation. However, but vertebral compression fracture (VCF) can be problematic. The aim of his study is to evaluate the outcome and risk of VCF after SABR for spinal metastasis from HCC. We retrospectively reviewed 33 lesions in 42 spinal segments from 29 patients who received SABR with 1 fraction (16-20 Gy), or 3 fractions (18-45 Gy) from September 2009 to January 2015. The 1-year local control (LC) rate was 68.3%. Radiographic grade of cord compression (RGCC) was the only independent prognostic factor associated with LC (P = 0.007). The 1-year ultimate LC rate including the outcome of salvage re-irradiation was 87.2%. The pain response rate was 73.3% according to the categories of the International Bone Metastases Consensus Group. The 1-year VCF-free rate was 71.5%. Pre-existing VCF (P < 0.001) and only-lytic change (P = 0.017) were associated with a higher post-SABR VCF rate. One-third of post-SABR VCFs required interventions. SABR for spinal metastases from HCC provided efficacious LC, especially for lesions with RGCC ≤ II, and showed effective and durable pain relief. As VCF after SABR occurred frequently for vertebral segments with pre-existing VCF and only-lytic change, early preventive vertebroplasty is considerable for those high-risk vertebral segments.

LFP-guided targeting of a cortical barrel column for in vivo two-photon calcium imaging.

Two-photon microscopy of bulk-loaded functional dyes is an outstanding physiological technique that enables simultaneous functional mapping of hundreds of brain cells in vivo at single-cell resolution. However, precise targeting of a specific cortical location is not easy due to its fine dimensionality. To enable precise targeting, intrinsic-signal optical imaging is often additionally performed. However, the intrinsic-signal optical imaging is not only time-consuming but also ineffective in ensuring precision. Here, we propose an alternative method for precise targeting based on local field potential (LFP) recording, a conventional electrophysiological method. The heart of this method lies in use of the same glass pipette to record LFPs and to eject calcium dye. After confirming the target area by LFP using a glass pipette, the calcium dye is ejected from the same pipette without a time delay or spatial adjustment. As a result, the calcium dye is loaded into the same ensemble of brain cells from which the LFP was obtained. As a validation of the proposed LFP-based method, we targeted and successfully loaded calcium dye into layer 2/3 of a mouse barrel column.

Changes in the activity of the muscles surrounding the neck according to the angles of movement of the neck in adults in their 20s.

[Purpose] The aim of this study was to examine changes in the muscle activity around the neck according to the neck movement angle during neck flexion and extension. [Subjects and Methods] Activities of the sternocleidomastoid muscle (SCM), splenius capitis and splenius cervicis muscles, upper trapezius muscle, and middle trapezius muscle during flexion and extension were assessed in 24 college students. [Results] SCM muscle activation significantly increased at every angle during flexion and extension. The activities of the splenius capitis and splenius cervicis muscles increased significantly during flexion. The activity of the upper trapezius muscle also increased significantly. [Conclusion] The results highlight the need for individuals not to adopt a neck flexion posture for extended periods.

Scheduled interval trans-catheter arterial chemoembolization followed by radiation therapy in patients with unresectable hepatocellular carcinoma.

Combination treatment of trans-catheter arterial chemoembolization (TACE) and conformal radiation therapy (RT) reported promising results in patients with hepatocellular carcinoma (HCC), but, optimal interval was not determined. We hypothesized that a two-week interval between TACE and RT would be optimal. Therefore, we designed this study to evaluate the safety and efficacy of scheduled interval TACE followed by RT. HCC patients who were not eligible for standard therapies were enrolled for scheduled interval TACE followed by RT (START). Patients received TACE on the first day of treatment, and then RT was delivered after 14 days. The entire course of treatment took between four and five weeks. In 81 patients (96.4%), START was completed in the planned treatment period. RT was delayed in the remaining three patients because of decreased liver function or poor performance status after TACE. Of the 81 patients, objective response was observed in 57 patients (70.4%). One unexpected death occurred after START due to hepatic failure. Other toxicities were manageable. The median survival was 14.7 months. There was a significant difference in overall survival according to the response to START (P < 0.001). In conclusion, START is safe and feasible.

Analysis of prognostic factors and 5-year survival rate in patients with hepatocellular carcinoma: a single-center experience.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC), which is the third most common cancer in Korea, has a very poor prognosis. However, only a few studies have performed a comprehensive survival-related analysis in all patients who were consecutively diagnosed and treated over a given period of time. The aim of this study was to determine the 5-year survival rate and its prognostic factors among HCC patients. METHODS: In total, 257 patients who were consecutively diagnosed with HCC between January 2000 and December 2003 were followed until death or until December 2008. We analyzed their survival outcomes according to their clinical characteristics, tumor staging, and treatment modalities, and determined the independent prognostic factors affecting survival. RESULTS: The patients were aged 59±10 years (mean±SD). During the follow-up period, 223 patients (86.8%) died and the overall median survival was 10.8 months; the 1-, 3-, and 5-year survival rates were 44.4%, 21.0%, and 12.1%, respectively. The outcomes in patients with tumor node metastasis (TNM) stage I or II and Child-Pugh class A or B were significantly better with surgical resection than with other treatment modalities (P<0.01). Patients who underwent supplementary transcatheter arterial chemoembolization as a second-line treatment after surgical resection had better outcomes than those who underwent surgical resection alone (P=0.02). Initial symptoms, Child-Pugh class, serum alpha-fetoprotein, tumor size, portal vein thrombosis, and TNM stage were found to be independent prognostic factors for survival among HCC patients. CONCLUSIONS: This retrospective cohort study elucidated survival outcomes and prognostic factors affecting survival in HCC patients at a single center.

Prognostic index for portal vein tumor thrombosis in patients with hepatocellular carcinoma treated with radiation therapy.

We performed a retrospective review of 281 hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) treated with radiation therapy (RT) between 1998 and 2008 to develop a prognostic model for those patients. Of the 281 patients, PVTT and intrahepatic main masses completely disappeared in 10 patients (3.6%), and shown a partial response in 141 patients (50.2%). The median survival was 11.6 months. Patients who had more than PR have shown significantly longer survival than the others (22.0 months vs 5.0 months, P < 0.001). On the multivariate analysis, pre-treatment poor prognosticators for overall survival were ECOG performance status, Child-Pugh class, multiple tumors, main PVTT, complete portal vein occlusion, lymph node metastasis, and primary tumor size. Prognostic index of RT for PVTT of HCC (PITH) scores were defined as the number of pre-treatment poor prognostic factors. PITH scores correlated well with overall survival. In the analysis of 1 and 2 yr overall survival rate, patients who had PITH scores of 3 or greater showed a significantly lower rate of overall survival than the others (33.0%, 17.3% vs 70.1%, 40.8%, respectively, P < 0.001). The PITH scoring model, proposed in the current study in HCC patients with PVTT, reliably predict overall survival.