VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

VEGF-C promotes brain-derived fluid drainage, confers neuroprotection, and improves stroke outcomes.

Meningeal lymphatic vessels promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelium growth factor-C (VEGF-C) is essential for meningeal lymphatic development and maintenance and has therapeutic potential for treating neurological disorders, including ischemic stroke. We have investigated the effects of VEGF-C overexpression on brain fluid drainage, single cell transcriptome in the brain, and stroke outcomes in adult mice. Intra-cerebrospinal fluid administration of an adeno-associated virus expressing VEGF-C (AAV-VEGF-C) increases the CNS lymphatic network. Post-contrast T1 mapping of the head and neck showed that deep cervical lymph node size and drainage of CNS-derived fluids were increased. Single nuclei RNA sequencing revealed a neuro-supportive role of VEGF-C via upregulation of calcium and brain-derived neurotrophic factor (BDNF) signaling pathways in brain cells. In a mouse model of ischemic stroke, AAV-VEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage. AAV-VEGF-C thus promotes CNS-derived fluid and solute drainage, confers neuroprotection, and reduces ischemic stroke damage. Short abstract: Intrathecal delivery of VEGF-C increases the lymphatic drainage of brain-derived fluids confers neuroprotection, and improves neurological outcomes after ischemic stroke.

Cellular recovery after prolonged warm ischaemia of the whole body.

After cessation of blood flow or similar ischaemic exposures, deleterious molecular cascades commence in mammalian cells, eventually leading to their death1,2. Yet with targeted interventions, these processes can be mitigated or reversed, even minutes or hours post mortem, as also reported in the isolated porcine brain using BrainEx technology3. To date, translating single-organ interventions to intact, whole-body applications remains hampered by circulatory and multisystem physiological challenges. Here we describe OrganEx, an adaptation of the BrainEx extracorporeal pulsatile-perfusion system and cytoprotective perfusate for porcine whole-body settings. After 1 h of warm ischaemia, OrganEx application preserved tissue integrity, decreased cell death and restored selected molecular and cellular processes across multiple vital organs. Commensurately, single-nucleus transcriptomic analysis revealed organ- and cell-type-specific gene expression patterns that are reflective of specific molecular and cellular repair processes. Our analysis comprises a comprehensive resource of cell-type-specific changes during defined ischaemic intervals and perfusion interventions spanning multiple organs, and it reveals an underappreciated potential for cellular recovery after prolonged whole-body warm ischaemia in a large mammal.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

AgRP neurons control structure and function of the medial prefrontal cortex.

Hypothalamic agouti-related peptide and neuropeptide Y-expressing (AgRP) neurons have a critical role in both feeding and non-feeding behaviors of newborn, adolescent, and adult mice, suggesting their broad modulatory impact on brain functions. Here we show that constitutive impairment of AgRP neurons or their peripubertal chemogenetic inhibition resulted in both a numerical and functional reduction of neurons in the medial prefrontal cortex (mPFC) of mice. These changes were accompanied by alteration of oscillatory network activity in mPFC, impaired sensorimotor gating, and altered ambulatory behavior that could be reversed by the administration of clozapine, a non-selective dopamine receptor antagonist. The observed AgRP effects are transduced to mPFC in part via dopaminergic neurons in the ventral tegmental area and may also be conveyed by medial thalamic neurons. Our results unmasked a previously unsuspected role for hypothalamic AgRP neurons in control of neuronal pathways that regulate higher-order brain functions during development and in adulthood.

Translator: A Transfer Learning Approach to Facilitate Single-Cell ATAC-Seq Data Analysis from Reference Dataset.

Recent advances in single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) have allowed simultaneous epigenetic profiling over thousands of individual cells to dissect the cellular heterogeneity and elucidate regulatory mechanisms at the finest possible resolution. However, scATAC-seq is challenging to model computationally due to the ultra-high dimensionality, low signal-to-noise ratio, complex feature interactions, and high vulnerability to various confounding factors. In this study, we present Translator, an efficient transfer learning approach to capture generalizable chromatin interactions from high-quality (HQ) reference scATAC-seq data to obtain robust cell representations in low-to-moderate quality target scATAC-seq data. We applied Translator on various simulated and real scATAC-seq datasets and demonstrated that Translator could learn more biologically meaningful cell representations than other methods by incorporating information learned from the reference data, thus facilitating various downstream analyses such as clustering and motif enrichment measurements. Moreover, Translator's block-wise deep learning framework can handle nonlinear relationships with restricted connections using fewer parameters to boost computational efficiency through Graphics Processing Unit (GPU) parallelism. Finally, we have implemented Translator as a free software package available for the community to leverage large-scale, HQ reference data to study target scATAC-seq data.

Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells.

The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer's disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer's disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.

NogoA-expressing astrocytes limit peripheral macrophage infiltration after ischemic brain injury in primates.

Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma.

Astrocyte Heterogeneity in Multiple Sclerosis: Current Understanding and Technical Challenges.

The emergence of single cell technologies provides the opportunity to characterize complex immune/central nervous system cell assemblies in multiple sclerosis (MS) and to study their cell population structures, network activation and dynamics at unprecedented depths. In this review, we summarize the current knowledge of astrocyte subpopulations in MS tissue and discuss the challenges associated with resolving astrocyte heterogeneity with single-nucleus RNA-sequencing (snRNA-seq). We further discuss multiplexed imaging techniques as tools for defining population clusters within a spatial context. Finally, we will provide an outlook on how these technologies may aid in answering unresolved questions in MS, such as the glial phenotypes that drive MS progression and/or neuropathological differences between different clinical MS subtypes.

Restoration of brain circulation and cellular functions hours post-mortem.

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Spatiotemporal transcriptomic divergence across human and macaque brain development.

Human nervous system development is an intricate and protracted process that requires precise spatiotemporal transcriptional regulation. We generated tissue-level and single-cell transcriptomic data from up to 16 brain regions covering prenatal and postnatal rhesus macaque development. Integrative analysis with complementary human data revealed that global intraspecies (ontogenetic) and interspecies (phylogenetic) regional transcriptomic differences exhibit concerted cup-shaped patterns, with a late fetal-to-infancy (perinatal) convergence. Prenatal neocortical transcriptomic patterns revealed transient topographic gradients, whereas postnatal patterns largely reflected functional hierarchy. Genes exhibiting heterotopic and heterochronic divergence included those transiently enriched in the prenatal prefrontal cortex or linked to autism spectrum disorder and schizophrenia. Our findings shed light on transcriptomic programs underlying the evolution of human brain development and the pathogenesis of neuropsychiatric disorders.

Expression and immunolocalization of metallothioneins MT1, MT2 and MT3 in rat nephron.

Rodent kidneys exhibit three isoforms of metallothioneins (MTs), MT1, MT2 and MT3, with poorly characterized localization along the nephron. Here we studied in adult male Wistar rats the renal expression of MTs mRNA by end-point RT-PCR and MT proteins by immunochemical methods The expression pattern of MT1 mRNA was cortex (CO)>outer stripe (OS)=inner stripe (IS)=inner medulla (IM), of MT2 mRNA was IM>CO>IS=OS, and of MT3 mRNA was IM>CO=OS=IM. MT1/2-antibody stained with heterogeneous intensity the cell cytoplasm and nuclei in proximal tubule (PT) and thin ascending limb, whereas MT3-antibody stained weakly the cell cytoplasm in various cortical tubules and strongly the nuclei in all nephron segments. However, the isolated nuclei exhibited an absence of MT1/2 and presence of MT3 protein. In MT1/2-positive PT cells, the intracellular staining appeared diffuse or bipolar, but the isolated brush-border, basolateral and endosomal membranes were devoid of MT1/2 proteins. In the lumen of some PT profiles, the heterogeneously sized MT1/2-rich vesicles were observed, with the limiting membrane positive for NHE3, but negative for V-ATPase, CAIV, and megalin, whereas their interior was positive for CAII and negative for cytoskeleton. They seem to be pinched off from the luminal membrane of MT1/2-rich cells, as also indicated by transmission electron microscopy. We conclude that in male rats, MTs are heterogeneously abundant in the cell cytoplasm and/or nuclei along the nephron. The MT1/2-rich vesicles in the tubule lumen may represent a source of urine MT and membranous material, whereas MT3 in nuclei may handle zink and locally-produced reactive oxygen species.

Molecular and cellular reorganization of neural circuits in the human lineage.

To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type-specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.

Modulation of inflammatory responses by a cannabinoid-2-selective agonist after spinal cord injury.

The goal of the current investigation was to evaluate the mechanisms through which administration of a selective cannabinoid-2 (CB2) agonist (O-1966) modifies inflammatory responses and helps to improve function following spinal cord injury. A comparison of motor function, autonomic function, and inflammatory responses was made between animals treated with O-1966 (5 mg/kg IP) and animals treated with vehicle 1 h and 24 h following contusion injury to the spinal cord. Motor function was significantly improved in the treated animals at each time point during the 14 days of evaluation. The percentage of animals able to spontaneously void their bladder was also greater over the entire study period in the group treated with the selective CB2 agonist. Seven days following injury there was a significant reduction in both hematopoietic and myeloid cell invasion of the spinal cord, and a reduction in the number of immunoreactive microglia. The results of the evaluation of chemokine/cytokine expression and inflammatory cell invasion also demonstrated a significant effect of treatment on inflammatory reactions following injury. Two days after injury, animals treated with O-1966 had significant reductions in CXCL-9 and CXCL-11, and dramatic reductions in IL-23p19 expression and its receptor IL-23r. Treatment with O-1966 also caused inhibition of toll-like receptor expression (TLR1, TLR4, TLR6 and TLR7) following injury. These results demonstrate that the improvement in motor and autonomic function resulting from treatment with a selective CB2 agonist is associated with a significant effect on inflammatory responses in the spinal cord following injury.

Novel mechanisms of immune modulation of natalizumab in multiple sclerosis patients.

The goal of this study was to investigate the effects of natalizumab therapy on the immune cell composition and phenotype in the blood of relapsing MS patients treated over the course of 12 months. We collected peripheral blood from 26 RRMS patients before treatment onset, and then 6 and 12 months after therapy. PBMC was isolated and then analyzed for phenotypic characteristics by FACS and for cytokine production by ELISA. The results of our studies showed changes in both numbers and activation states of immune cells following therapy. These changes were observed at the 6 month timepoint and generally persisted through the 12 month timepoint. The proportions of NK cells (CD3⁻CD56+) and hematopoetic stem cells (CD34+lin⁻) were increased after natalizumab treatment. Decreases were noted in numbers of CD14+ monocytes, and possibly their migratory potential, since their expression levels of α4ß1 were decreased. Relative numbers of CD20+ B cells were increased, but the proportion of CD20+ cells expressing high levels of α4ß1 integrin was decreased. While proportions of CD4+ and CD8+ T cells did not change, the percentage of cells expressing α4ß1 integrin was significantly decreased for both subsets. Natalizumab therapy produces a number of phenotypic changes in the immune composition of peripheral blood. These changes may help to explain both the mechanisms of action of natalizumab and also shed light on the potential for the observed increase in PML in these patients.

Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs.

Metallothioneins are cysteine-rich, small metal-binding proteins present in various mammalian tissues. Of the four common metallothioneins, MT-1 and MT-2 (MTs) are expressed in most tissues, MT-3 is predominantly present in brain, whereas MT-4 is restricted to the squamous epithelia. The expression of MT-1 and MT-2 in some organs exhibits sex, age, and strain differences, and inducibility with a variety of stimuli. In adult mammals, MTs have been localized largely in the cell cytoplasm, but also in lysosomes, mitochondria and nuclei. The major physiological functions of MTs include homeostasis of essential metals Zn and Cu, protection against cytotoxicity of Cd and other toxic metals, and scavenging free radicals generated in oxidative stress. The role of MTs in Cd-induced acute and chronic toxicity, particularly in liver and kidneys, is reviewed in more details. In acute toxicity, liver is the primary target, whereas in chronic toxicity, kidneys are major targets of Cd. The intracellular MTs bind Cd ions and form CdMT. In chronic intoxication, Cd stimulates de novo synthesis of MTs; it is assumed that toxicity in the cells starts when loading with Cd ions exceeds the buffering capacity of intracellular MTs. CdMT, released from the Cd-injured organs, or when applied parenterally for experimental purposes, reaches the kidneys via circulation, where it is filtered, endocytosed in the proximal tubule cells, and degraded in lysosomes. Liberated Cd can immediately affect the cell structures and functions. The resulting proteinuria and CdMT in the urine can be used as biomarkers of tubular injury.

Dendritic cells transduced with lentiviral vectors expressing VIP differentiate into VIP-secreting tolerogenic-like DCs.

Dendritic cells (DCs) initiate immune responses as well as tolerance. We showed previously that the neuropeptide vasoactive intestinal peptide (VIP) suppresses innate immune responses, modulates adaptive responses by generating regulatory T cells (Treg) through the induction of tolerogenic DCs (tDCs), and has therapeutic effects in models of autoimmune/inflammatory disorders. Systemic VIP administration is limited by its short biological half-life and by its pleiotropic effects on the cardiovascular system and gastrointestinal tract. Therefore, we used lentiviral vectors to genetically engineer VIP-expressing bone marrow-derived DC (BMDC) and characterized the transduced LentiVIP-DC in terms of phenotype and therapeutic effects in models of experimental autoimmune encephalomyelitis (EAE) and cecal ligation and puncture (CLP) sepsis. LentiVIP-DCs secrete VIP, and resemble tDCs through lack of co-stimulatory molecule upregulation, lack of proinflammatory cytokine secretion, increased interleukin (IL)-10 production, and poor stimulation of allogeneic T cells. A single inoculation of LentiVIP-DC in EAE or CLP mice had therapeutic effects, which correlated with reduced expression of proinflammatory cytokines and increased IL-10 production in spinal cord and peritoneal fluid, respectively. In contrast to systemic VIP administration that requires repeated, high-dose inoculations, local delivery of VIP by LentiVIP-DC may represent a promising therapeutic tool for the treatment of autoimmune diseases and inflammatory disorders.

Cyclophosphamide resets dendritic cell homeostasis and enhances antitumor immunity through effects that extend beyond regulatory T cell elimination.

Using a model of established malignancy, we found that cyclophosphamide (Cy), administered at a dose not requiring hematopoietic stem cell support, is superior to low-dose total body irradiation in augmenting antitumor immunity. We observed that Cy administration resulted in expansion of tumor antigen-specific T cells and transient depletion of CD4(+)Foxp3(+) regulatory T cells (Tregs). The antitumor efficacy of Cy was not improved by administration of anti-CD25 monoclonal antibody given to induce more profound Treg depletion. We found that Cy, through its myelosuppressive action, induced rebound myelopoiesis and perturbed dendritic cell (DC) homeostasis. The resulting DC turnover led to the emergence of tumor-infiltrating DCs that secreted more IL-12 and less IL-10 compared to those from untreated tumor-bearing animals. These newly recruited DCs, originating from proliferating early DC progenitors, were fully capable of priming T cell responses and ineffective in inducing expansion of Tregs. Together, our results show that Cy-mediated antitumor effects extend beyond the well-documented cytotoxicity and lymphodepletion and include resetting the DC homeostasis, thus providing an excellent platform for integration with other immunotherapeutic strategies.

Signal transduction inhibition of APCs diminishes th17 and Th1 responses in experimental autoimmune encephalomyelitis.

IL-17- and IFN-gamma-secreting T cells play an important role in autoimmune responses in multiple sclerosis and the model system experimental autoimmune encephalomyelitis (EAE). Dendritic cells (DCs) in the periphery and microglia in the CNS are responsible for cytokine polarization and expansion of this T cell subset. Our results indicate that in vivo administration of a signal transduction inhibitor that targets DCs to mice with EAE led to a decrease in CNS infiltration of pathogenic Ag-specific T cells. Since this approach does not target T cells directly, we assessed the effects on the APCs that are involved in generating the T cell responses. Since in EAE and multiple sclerosis, both microglia and peripheral DCs are likely to contribute to disease, we utilized a bone marrow chimera system to distinguish between these two populations. These studies show that peripheral DCs are the primary target but that microglia are also modestly affected by CEP-701, as numbers and activation states of the cells in the CNS are decreased after therapy. Our results also showed a decrease in secretion of TNF-alpha, IL-6, and IL-23 by DCs as well as a decrease in expression of costimulatory molecules. We further determined that levels of phospho-Stat1, Stat3, Stat5, and NF-kappaB, which are signaling molecules that have been implicated in these pathways, were decreased. Thus, use of this class of signal transduction inhibitors may represent a novel method to treat autoimmunity by dampening the autoreactive polarizing condition driven by DCs.

Factors governing the activation of adoptively transferred donor T cells infused after allogeneic bone marrow transplantation in the mouse.

Murine models of bone marrow transplantation were used to study the mechanisms governing the activation of donor lymphocyte infusions (DLIs) manifesting as lymphohematopoietic graft-versus-host (LH-GVH) and graft-versus-leukemia (GVL) reactivities. We demonstrate here that established mixed chimerism influences the potency of DLI-mediated alloreactivity only in the MHC-mismatched but not MHC-matched setting. In the MHC-matched setting, high levels (>or= 40%) of residual host chimerism correlated negatively with DLI-mediated alloreactivity irrespective of the timing of their administration, the donor's previous sensitization to host antigens, or the level of residual host APCs. In vivo administration of Toll-like receptor (TLR) ligands was required to maximize DLI-mediated LH-GVH and GVL reactivities in chimeras with low levels (