CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Structural and signaling mechanisms of TAAR1 enabled preferential agonist design.

Trace amine-associated receptor 1 (TAAR1) senses a spectrum of endogenous amine-containing metabolites (EAMs) to mediate diverse psychological functions and is useful for schizophrenia treatment without the side effects of catalepsy. Here, we systematically profiled the signaling properties of TAAR1 activation and present nine structures of TAAR1-Gs/Gq in complex with EAMs, clinical drugs, and synthetic compounds. These structures not only revealed the primary amine recognition pocket (PARP) harboring the conserved acidic D3.32 for conserved amine recognition and "twin" toggle switch for receptor activation but also elucidated that targeting specific residues in the second binding pocket (SBP) allowed modulation of signaling preference. In addition to traditional drug-induced Gs signaling, Gq activation by EAM or synthetic compounds is beneficial to schizophrenia treatment. Our results provided a structural and signaling framework for molecular recognition by TAAR1, which afforded structural templates and signal clues for TAAR1-targeted candidate compounds design.

Differentiation route determines the functional outputs of adult megakaryopoiesis.

Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.

A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos.

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Dual Antiplatelet Treatment up to 72 Hours after Ischemic Stroke.

BACKGROUND: Dual antiplatelet treatment has been shown to lower the risk of recurrent stroke as compared with aspirin alone when treatment is initiated early (≤24 hours) after an acute mild stroke. The effect of clopidogrel plus aspirin as compared with aspirin alone administered within 72 hours after the onset of acute cerebral ischemia from atherosclerosis has not been well studied. METHODS: In 222 hospitals in China, we conducted a double-blind, randomized, placebo-controlled, two-by-two factorial trial involving patients with mild ischemic stroke or high-risk transient ischemic attack (TIA) of presumed atherosclerotic cause who had not undergone thrombolysis or thrombectomy. Patients were randomly assigned, in a 1:1 ratio, within 72 hours after symptom onset to receive clopidogrel (300 mg on day 1 and 75 mg daily on days 2 to 90) plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 21) or matching clopidogrel placebo plus aspirin (100 to 300 mg on day 1 and 100 mg daily on days 2 to 90). There was no interaction between this component of the factorial trial design and a second part that compared immediate with delayed statin treatment (not reported here). The primary efficacy outcome was new stroke, and the primary safety outcome was moderate-to-severe bleeding - both assessed within 90 days. RESULTS: A total of 6100 patients were enrolled, with 3050 assigned to each trial group. TIA was the qualifying event for enrollment in 13.1% of the patients. A total of 12.8% of the patients were assigned to a treatment group no more than 24 hours after stroke onset, and 87.2% were assigned after 24 hours and no more than 72 hours after stroke onset. A new stroke occurred in 222 patients (7.3%) in the clopidogrel-aspirin group and in 279 (9.2%) in the aspirin group (hazard ratio, 0.79; 95% confidence interval [CI], 0.66 to 0.94; P = 0.008). Moderate-to-severe bleeding occurred in 27 patients (0.9%) in the clopidogrel-aspirin group and in 13 (0.4%) in the aspirin group (hazard ratio, 2.08; 95% CI, 1.07 to 4.04; P = 0.03). CONCLUSIONS: Among patients with mild ischemic stroke or high-risk TIA of presumed atherosclerotic cause, combined clopidogrel-aspirin therapy initiated within 72 hours after stroke onset led to a lower risk of new stroke at 90 days than aspirin therapy alone but was associated with a low but higher risk of moderate-to-severe bleeding. (Funded by the National Natural Science Foundation of China and others; INSPIRES ClinicalTrials.gov number, NCT03635749.).

Trial of Endovascular Therapy for Acute Ischemic Stroke with Large Infarct.

BACKGROUND: The role of endovascular therapy for acute stroke with a large infarction has not been extensively studied in differing populations. METHODS: We conducted a multicenter, prospective, open-label, randomized trial in China involving patients with acute large-vessel occlusion in the anterior circulation and an Alberta Stroke Program Early Computed Tomography Score of 3 to 5 (range, 0 to 10, with lower values indicating larger infarction) or an infarct-core volume of 70 to 100 ml. Patients were randomly assigned in a 1:1 ratio within 24 hours from the time they were last known to be well to undergo endovascular therapy and receive medical management or to receive medical management alone. The primary outcome was the score on the modified Rankin scale at 90 days (scores range from 0 to 6, with higher scores indicating greater disability), and the primary objective was to determine whether a shift in the distribution of the scores on the modified Rankin scale at 90 days had occurred between the two groups. Secondary outcomes included scores of 0 to 2 and 0 to 3 on the modified Rankin scale. The primary safety outcome was symptomatic intracranial hemorrhage within 48 hours after randomization. RESULTS: A total of 456 patients were enrolled; 231 were assigned to the endovascular-therapy group and 225 to the medical-management group. Approximately 28% of the patients in both groups received intravenous thrombolysis. The trial was stopped early owing to the efficacy of endovascular therapy after the second interim analysis. At 90 days, a shift in the distribution of scores on the modified Rankin scale toward better outcomes was observed in favor of endovascular therapy over medical management alone (generalized odds ratio, 1.37; 95% confidence interval, 1.11 to 1.69; P = 0.004). Symptomatic intracranial hemorrhage occurred in 14 of 230 patients (6.1%) in the endovascular-therapy group and in 6 of 225 patients (2.7%) in the medical-management group; any intracranial hemorrhage occurred in 113 (49.1%) and 39 (17.3%), respectively. Results for the secondary outcomes generally supported those of the primary analysis. CONCLUSIONS: In a trial conducted in China, patients with large cerebral infarctions had better outcomes with endovascular therapy administered within 24 hours than with medical management alone but had more intracranial hemorrhages. (Funded by Covidien Healthcare International Trading [Shanghai] and others; ANGEL-ASPECT ClinicalTrials.gov number, NCT04551664.).

Trichinella spiralis -induced immunomodulation signatures on gut microbiota and metabolic pathways in mice.

The hygiene hypothesis proposes that decreased exposure to infectious agents in developed countries may contribute to the development of allergic and autoimmune diseases. Trichinella spiralis, a parasitic roundworm, causes trichinellosis, also known as trichinosis, in humans. T. spiralis had many hosts, and almost any mammal could become infected. Adult worms lived in the small intestine, while the larvae lived in muscle cells of the same mammal. T. spiralis was a significant public health threat because it could cause severe illness and even death in humans who eat undercooked or raw meat containing the parasite. The complex interactions between gastrointestinal helminths, gut microbiota, and the host immune system present a challenge for researchers. Two groups of mice were infected with T. spiralis vs uninfected control, and the experiment was conducted over 60 days. The 16S rRNA gene sequences and untargeted LC/MS-based metabolomics of fecal and serum samples, respectively, from different stages of development of the Trichinella spiralis-mouse model, were examined in this study. Gut microbiota alterations and metabolic activity accompanied by parasite-induced immunomodulation were detected. The inflammation parameters of the duodenum (villus/crypt ratio, goblet cell number and size, and histological score) were involved in active inflammation and oxidative metabolite profiles. These profiles included increased biosynthesis of phenylalanine, tyrosine, and tryptophan while decreasing cholesterol metabolism and primary and secondary bile acid biosynthesis. These disrupted metabolisms adapted to infection stress during the enteral and parenteral phases and then return to homeostasis during the encapsulated phase. There was a shift from an abundance of Bacteroides in the parenteral phase to an abundance of probiotic Lactobacillus and Treg-associated-Clostridia in the encapsulated phase. Th2 immune response (IL-4/IL-5/IL-13), lamina propria Treg, and immune hyporesponsiveness metabolic pathways (decreased tropane, piperidine and pyridine alkaloid biosynthesis and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid) were all altered. These findings enhanced our understanding of gut microbiota and metabolic profiles of Trichinella -infected mice, which could be a driving force in parasite-shaping immune system maintenance.

Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord.

K+-Cl- cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In Cacna2d1 KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.Significance statementImpaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1­NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.

Calcineurin and CK2 Reciprocally Regulate Synaptic AMPA Receptor Phenotypes via α2δ-1 in Spinal Excitatory Neurons.

Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these drugs can cause severe and persistent pain. GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) are implicated in various neurological disorders, including neuropathic pain. It is unclear whether and how constitutive calcineurin, a Ca2+/calmodulin protein phosphatase, controls synaptic CP-AMPARs. In this study, we found that blocking CP-AMPARs with IEM-1460 markedly reduced the amplitude of AMPAR-EPSCs in excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2), but not in inhibitory neurons expressing vesicular GABA transporter, in the spinal cord of FK506-treated male and female mice. FK506 treatment also caused an inward rectification in the current-voltage relationship of AMPAR-EPSCs specifically in VGluT2 neurons. Intrathecal injection of IEM-1460 rapidly alleviated pain hypersensitivity in FK506-treated mice. Furthermore, FK506 treatment substantially increased physical interaction of α2δ-1 with GluA1 and GluA2 in the spinal cord and reduced GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of spinal cords. Correspondingly, inhibiting α2δ-1 with pregabalin, Cacna2d1 genetic knock-out, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide reversed inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons caused by FK506 treatment. In addition, CK2 inhibition reversed FK506 treatment-induced pain hypersensitivity, α2δ-1 interactions with GluA1 and GluA2, and inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons. Thus, the increased prevalence of synaptic CP-AMPARs in spinal excitatory neurons plays a major role in calcineurin inhibitor-induced pain hypersensitivity. Calcineurin and CK2 antagonistically regulate postsynaptic CP-AMPARs through α2δ-1-mediated GluA1/GluA2 heteromeric assembly in the spinal dorsal horn.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Alternative Splicing Factor Heterogeneous Nuclear Ribonucleoprotein U as a Promising Biomarker for Gastric Cancer Risk and Prognosis with Tumor-Promoting Properties.

Gastric cancer (GC) is a major global health concern with poor outcomes. Heterogeneous nuclear ribonucleoprotein U (HNRNPU) is a multifunctional protein that participates in pre-mRNA packaging, alternative splicing regulation, and chromatin remodeling. Its potential role in GC remains unclear. In this study, the expression characteristics of HNRNPU were analyzed by The Cancer Genome Atlas data, Gene Expression Omnibus data, and then further identified by real-time quantitative PCR and immunohistochemistry using tissue specimens. From superficial gastritis, atrophic gastritis, and hyperplasia to GC, the in situ expression of HNRNPU protein gradually increased, and the areas under the curve for diagnosis of GC and its precancerous lesions were 0.911 and 0.847, respectively. A nomogram integrating HNRNPU expression, lymph node metastasis, and other prognostic indicators exhibited an area under the curve of 0.785 for predicting survival risk. Knockdown of HNRNPU significantly inhibited GC cell proliferation, migration, and invasion and promoted apoptosis in vitro. In addition, RNA-sequencing analysis showed that HNRNPU could affect alternative splicing events in GC cells, with functional enrichment analysis revealing that HNRNPU may exert malignant biological function in GC progression through alternative splicing regulation. In summary, the increased expression of HNRNPU was significantly associated with the development of GC, with a good performance in diagnosing and predicting the prognostic risk of GC. Functionally, HNRNPU may play an oncogenic role in GC by regulating alternative splicing.

Dalbergia odorifera undergoes massive molecular shifts in response to waterlogging combined with salinity.

Field and greenhouse studies attempting to describe the molecular responses of plant species under waterlogging (WL) combined with salinity (ST) are almost nonexistent. We integrated transcriptional, metabolic, and physiological responses involving several crucial transcripts and common differentially expressed genes and metabolites in fragrant rosewood (Dalbergia odorifera) leaflets to dissect plant-specific molecular responses and patterns under WL combined with ST (SWL). We discovered that the synergistic pattern of the transcriptional response of fragrant rosewood under SWL was exclusively characterized by the number of regulated transcripts. The response patterns under SWL based on transcriptome and metabolome regulation statuses revealed different patterns (additive, dominant, neutral, minor, unilateral, and antagonistic) of transcripts or metabolites that were commonly regulated or expressed uniquely under SWL. Under SWL, the synergistic transcriptional response of several functional gene subsets was positively associated with several metabolomic and physiological responses related to the shutdown of the photosynthetic apparatus and the extensive degradation of starch into saccharides through α-amylase, ß-amylase, and α-glucosidase or plastoglobuli accumulation. The dissimilarity between the regulation status and number of transcripts in plants under combined stresses led to nonsynergistic responses in several physiological and phytohormonal traits. As inferred from the impressive synergistic transcriptional response to morpho-physiological changes, combined stresses exhibited a gradually decreasing effect on the changes observed at the molecular level compared to those in the morphological one. Here, by characterizing the molecular responses and patterns of plant species under SWL, our study considerably improves our understanding of the molecular mechanisms underlying combined stress.

Cortical Microhemorrhage Presentation of Small Vessel Primary Angiitis of the Central Nervous System.

OBJECTIVE: Primary angiitis of the central nervous system (PACNS) is a rare vasculitis restricted to the brain, spinal cord, and leptomeninges. This study aimed to describe the imaging characteristics of patients with small vessel PACNS (SV-PACNS) using 7 T magnetic resonance imaging (MRI). METHODS: This ongoing prospective observational cohort study included patients who met the Calabrese and Mallek criteria and underwent 7 T MRI scan. The MRI protocol includes T1-weighted magnetization-prepared rapid gradient echo imaging, T2 star weighted imaging, and susceptibility-weighted imaging. Two experienced readers independently reviewed the neuroimages. Clinical data were extracted from the electronic patient records. The findings were then applied to a cohort of patients with large vessel central nervous system (CNS) vasculitis. RESULTS: We included 21 patients with SV-PACNS from December 2021 to November 2023. Of these, 12 (57.14%) had cerebral cortical microhemorrhages with atrophy. The pattern with microhemorrhages was described in detail based on the gradient echo sequence, leading to the identification of what we have termed the "coral-like sign." The onset age of patients with coral-like sign (33.83 ± 9.93 years) appeared younger than that of patients without coral-like sign (42.11 ± 14.18 years) (P = 0.131). Furthermore, the cerebral lesions in patients with cortical microhemorrhagic SV-PACNS showed greater propensity toward bilateral lesions (P = 0.03). The coral-like sign was not observed in patients with large vessel CNS vasculitis. INTERPRETATION: The key characteristics of the coral-like sign represent cerebral cortical diffuse microhemorrhages with atrophy, which may be an important MRI pattern of SV-PACNS. ANN NEUROL 2024;96:194-203.

Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Brain α2δ-1-Bound NMDA Receptors Drive Calcineurin Inhibitor-Induced Hypertension.

BACKGROUND: Calcineurin is highly enriched in immune T cells and the nervous system. Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), are the cornerstone of immunosuppressive regimens for preserving transplanted organs and tissues. However, these drugs often cause persistent hypertension owing to excess sympathetic outflow, which is maintained by N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input to the hypothalamic paraventricular nucleus (PVN). It is unclear how calcineurin inhibitors increase NMDAR activity in the PVN to augment sympathetic vasomotor activity. α2δ-1 (encoded by the Cacna2d1 gene), known colloquially as a calcium channel subunit, is a newly discovered NMDAR-interacting protein. In this study, we determined whether α2δ-1 plays a role in calcineurin inhibitor-induced synaptic NMDAR hyperactivity in the PVN and hypertension development. METHODS: Immunoblotting and coimmunoprecipitation assays were used to quantify synaptic protein levels and the physical interaction between GluN1 (the obligatory NMDAR subunit) and α2δ-1. Whole-cell patch-clamp recordings of retrogradely labeled, spinally projecting PVN were conducted in perfused brain slices to measure presynaptic and postsynaptic NMDAR activity. Radio-telemetry was implanted in rodents to continuously record arterial blood pressure in conscious states. RESULTS: Prolonged treatment with FK506 in rats significantly increased protein levels of α2δ-1, GluN1, and the α2δ-1-GluN1 complex in PVN synaptosomes. These effects were blocked by inhibiting α2δ-1 with gabapentin or interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus peptide. Treatment with FK506 potentiated the activity of presynaptic and postsynaptic NMDARs in spinally projecting PVN neurons; such effects were abolished by gabapentin, Cacna2d1 knockout, or α2δ-1 C-terminus peptide. Furthermore, microinjection of α2δ-1 C-terminus peptide into the PVN diminished renal sympathetic nerve discharges and arterial blood pressure that had been increased by FK506 treatment. Remarkably, concurrent administration of gabapentin prevented the development of FK506-induced hypertension in rats. Additionally, FK506 treatment induced sustained hypertension in wild-type mice but not in Cacna2d1 knockout mice. CONCLUSIONS: α2δ-1 is essential for calcineurin inhibitor-induced increases in synaptic NMDAR activity in PVN presympathetic neurons and sympathetic outflow. Thus, α2δ-1 and α2δ-1-bound NMDARs represent new targets for treating calcineurin inhibitor-induced hypertension. Gabapentinoids (gabapentin and pregabalin) could be repurposed for treating calcineurin inhibitor-induced neurogenic hypertension.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

m6A epitranscriptomic and epigenetic crosstalk in cardiac fibrosis.

Cardiac fibrosis, a crucial pathological characteristic of various cardiac diseases, presents a significant treatment challenge. It involves the deposition of the extracellular matrix (ECM) and is influenced by genetic and epigenetic factors. Prior investigations have predominantly centered on delineating the substantial influence of epigenetic and epitranscriptomic mechanisms in driving the progression of fibrosis. Recent studies have illuminated additional avenues for modulating the progression of fibrosis, offering potential solutions to the challenging issues surrounding fibrosis treatment. In the context of cardiac fibrosis, an intricate interplay exists between m6A epitranscriptomic and epigenetics. This interplay governs various pathophysiological processes: mitochondrial dysfunction, mitochondrial fission, oxidative stress, autophagy, apoptosis, pyroptosis, ferroptosis, cell fate switching, and cell differentiation, all of which affect the advancement of cardiac fibrosis. In this comprehensive review, we meticulously analyze pertinent studies, emphasizing the interplay between m6A epitranscriptomics and partial epigenetics (including histone modifications and noncoding RNA), aiming to provide novel insights for cardiac fibrosis treatment.