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Microglia, the innate immune cells of the brain, influence Alzheimer’s disease (AD) progression and are potential therapeutic targets. However, microglia exhibit diverse functions, the regulation of which is not fully understood, complicating therapeutics development. To better define the transcriptomic phenotypes and gene regulatory networks associated with AD, we enriched for microglia nuclei from 12 AD and 10 control human dorsolateral prefrontal cortices (7 males and 15 females, all aged >60 years) before single-nucleus RNA sequencing. Here we describe both established and previously unrecognized microglial molecular phenotypes, the inferred gene networks driving observed transcriptomic change, and apply trajectory analysis to reveal the putative relationships between microglial phenotypes. We identify microglial phenotypes more prevalent in AD cases compared with controls. Further, we describe the heterogeneity in microglia subclusters expressing homeostatic markers. Our study demonstrates that deep profiling of microglia in human AD brain can provide insight into microglial transcriptional changes associated with AD.

Scalable technologies to sequence the transcriptomes and epigenomes of single cells are transforming our understanding of cell types and cell states. The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network (BICCN) is applying these technologies at unprecedented scale to map the cell types in the mammalian brain. In an effort to increase data FAIRness (Findable, Accessible, Interoperable, Reusable), the NIH has established repositories to make data generated by the BICCN and related BRAIN Initiative projects accessible to the broader research community. Here, we describe the Neuroscience Multi-Omic Archive (NeMO Archive; nemoarchive.org), which serves as the primary repository for genomics data from the BRAIN Initiative. Working closely with other BRAIN Initiative researchers, we have organized these data into a continually expanding, curated repository, which contains transcriptomic and epigenomic data from over 50 million brain cells, including single-cell genomic data from all of the major regions of the adult and prenatal human and mouse brains, as well as substantial single-cell genomic data from non-human primates. We make available several tools for accessing these data, including a searchable web portal, a cloud-computing interface for large-scale data processing (implemented on Terra, terra.bio), and a visualization and analysis platform, NeMO Analytics (nemoanalytics.org).

Scalable technologies to sequence the transcriptomes and epigenomes of single cells are transforming our understanding of cell types and cell states. The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network (BICCN) is applying these technologies at unprecedented scale to map the cell types in the mammalian brain. In an effort to increase data FAIRness (Findable, Accessible, Interoperable, Reusable), the NIH has established repositories to make data generated by the BICCN and related BRAIN Initiative projects accessible to the broader research community. Here, we describe the Neuroscience Multi-Omic Archive (NeMO Archive; nemoarchive.org), which serves as the primary repository for genomics data from the BRAIN Initiative. Working closely with other BRAIN Initiative researchers, we have organized these data into a continually expanding, curated repository, which contains transcriptomic and epigenomic data from over 50 million brain cells, including single-cell genomic data from all of the major regions of the adult and prenatal human and mouse brains, as well as substantial single-cell genomic data from non-human primates. We make available several tools for accessing these data, including a searchable web portal, a cloud-computing interface for large-scale data processing (implemented on Terra, terra.bio), and a visualization and analysis platform, NeMO Analytics (nemoanalytics.org).KEY POINTSThe Neuroscience Multi-Omic Archive serves as the genomics data repository for the BRAIN Initiative.Genomic data from >50 million cells span all the major regions of the brains of humans and mice.We provide a searchable web portal, a cloud-computing interface, and a data visualization platform.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro. To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo. Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion. SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Microglia are the innate immune cells of the central nervous system that adopt rapid functional changes in response to Damage Associated Molecular Patterns, including aggregated β-Amyloid (Aβ) found in Alzheimer's disease (AD). microRNAs (miRNAs) are post-transcriptional modulators that influence the timing and magnitude of microglia inflammatory responses by downregulating the expression of inflammatory effectors. Recent studies implicate miR-155, a miRNA known to regulate inflammatory responses, in the pathogenesis of neurodegenerative disorders including multiple sclerosis, ALS, familial Parkinson's disease, and AD. In this work, we asked if miR-155 expression in microglia modifies cellular behaviors in response to fibrillar Aβ1-42 (fAβ1-42 ), in vitro. We hypothesized that in microglia, miR-155 expression would impact the internalization and catabolism of extracellular fAβ1-42 . Primary microglia stimulated with lipopolysaccharide demonstrate fast upregulation of miR-155 followed by delayed upregulation of miR-146a, an anti-inflammatory miRNA. Conditional overexpression of miR-155 in microglia resulted in significant upregulation of miR-146a. Conditional deletion of miR-155 promoted transit of fAβ1-42 to low-pH compartments where catabolism occurs, while miR-155 overexpression decreases fAβ1-42 catabolism. Uptake of fAβ1-42 across the plasma membrane increased with both up and downregulation of miR-155 expression. Taken together, our results support the hypothesis that inflammatory signaling influences the ability of microglia to catabolize fAβ1-42 through interconnected mechanisms modulated by miR-155. Understanding how miRNAs modulate the ability of microglia to catabolize fAβ1-42 will further elucidate the role of cellular players and molecular crosstalk in AD pathophysiology.

Background Microglia have recently been implicated in opioid dependence and withdrawal. Mu Opioid (MOR) receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (MOR mediated) and indirect (neuron-interaction mediated) effects on microglia function. Methods To investigate this directly, we used RNA sequencing of ribosome-associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and followed by naloxone precipitated withdrawal (n=16). We validated the RNA-Seq data by combining fluorescent in-situ hybridization with immunohistochemistry for microglia (n=18). Finally, we expressed and activated the Gi/o-coupled hM4Di DREADD receptor in CX3CR1-expressing cells during morphine withdrawal (n=18). Results We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. WGCNA analysis revealed an intriguing network of cAMP-associated genes that are known to be involved in microglial motility, morphology, and interactions with neurons that were downregulated with morphine tolerance and upregulated rapidly by withdrawal. Three-dimensional histological reconstruction of microglia allowed for volumetric, visual colocalization of mRNA within individual microglia that validated our bioinformatics results. Direct activation of Gi/o-coupled DREADD receptors in CX3CR1-expressing cells exacerbated signs of opioid withdrawal rather than mimicking the effects of morphine. Conclusions These results indicate that Gi-signaling and cAMP-associated gene networks are inversely engaged during opioid tolerance and early withdrawal, perhaps revealing a role of microglia in mitigating the consequences of opioids.

Background: Early-onset familial Alzheimer disease (EOFAD) is caused by heterozygous variants in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP genes. Decades after their discovery, the mechanisms by which these genes cause Alzheimer’s disease (AD) or promote AD progression are not fully understood. While it is established that presenilin (PS) enzymatic activity produces amyloid-β (Aβ), PSs also regulate numerous other cellular functions, some of which intersect with known pathogenic drivers of neurodegeneration. Accumulating evidence suggests that microglia, resident innate immune cells in the central nervous system, play a key role in AD neurodegeneration. Objective: Previous work has identified a regulatory role for PS2 in microglia. We hypothesized that PSEN2 variants lead to dysregulated microglia, which could further contribute to disease acceleration. To mimic the genotype of EOFAD patients, we created a transgenic mouse expressing PSEN2 N141I on a mouse background expressing one wildtype PS2 and two PS1 alleles. Results: Microglial expression of PSEN2 N141I resulted in impaired γ-secretase activity as well as exaggerated inflammatory cytokine release, NFκB activity, and Aβ internalization. In vivo, PS2 N141I mice showed enhanced IL-6 and TREM2 expression in brain as well as reduced branch number and length, an indication of “activated” morphology, in the absence of inflammatory stimuli. LPS intraperitoneal injection resulted in higher inflammatory gene expression in PS2 N141I mouse brain relative to controls. Conclusion: Our findings demonstrate that PSEN2 N141I heterozygosity is associated with disrupted innate immune homeostasis, suggesting EOFAD variants may promote disease progression through non-neuronal cells beyond canonical dysregulated Aβ production.

Immunotherapy for haematologic malignancies with CD19-directed chimeric antigen receptor T cells has been highly successful at eradicating cancer but is associated with acute neurotoxicity in ∼40% of patients. This neurotoxicity correlates with systemic cytokine release syndrome, endothelial activation and disruption of endothelial integrity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We hypothesized that dysfunction of the neurovascular unit is a key step in the development of neurotoxicity. To recapitulate the interaction of the intact immune system with the blood–brain barrier, we first developed an immunocompetent mouse model of chimeric antigen receptor T-cell treatment-associated neurotoxicity. We treated wild-type mice with cyclophosphamide lymphodepletion followed by escalating doses of murine CD19-directed chimeric antigen receptor T cells. Within 3–5 days after chimeric antigen receptor T-cell infusion, these mice developed systemic cytokine release and abnormal behaviour as measured by daily neurologic screening exams and open-field testing. Histologic examination revealed widespread brain haemorrhages, diffuse extravascular immunoglobulin deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. To measure any associated changes in cerebral microvascular blood flow, we performed in vivo two-photon imaging through thinned-skull cranial windows. Unexpectedly, we found that 11.9% of cortical capillaries were plugged by Day 6 after chimeric antigen receptor T-cell treatment, compared to 1.1% in controls treated with mock transduced T cells. The capillary plugs comprised CD45+ leucocytes, a subset of which were CD3+ T cells. Plugging of this severity is expected to compromise cerebral perfusion. Indeed, we found widely distributed patchy hypoxia by hypoxyprobe immunolabelling. Increased serum levels of soluble ICAM-1 and VCAM-1 support a putative mechanism of increased leucocyte–endothelial adhesion. These data reveal that brain capillary obstruction may cause sufficient microvascular compromise to explain the clinical phenotype of chimeric antigen receptor T-cell neurotoxicity. The translational impact of this finding is strengthened by the fact that our mouse model closely approximates the kinetics and histologic findings of the chimeric antigen receptor T-cell neurotoxicity syndrome seen in human patients. This new link between systemic immune activation and neurovascular unit injury may be amenable to therapeutic intervention.

Microglia-mediated neuroinflammation is hypothesized to contribute to disease progression in neurodegenerative diseases such as Alzheimer9s Disease (AD). Microglia demonstrate heterogeneous states in health and disease, with proposed beneficial, harmful, and disease specific subtypes. Defining the spectrum of microglia phenotypes is an important step in rational design of neuroinflammation modulating therapies. To facilitate improved phenotype resolution and group comparisons based on disease state we performed single-nucleus RNA-seq on more than 120,000 microglia nuclei from AD and control dorsolateral prefrontal cortex. We identify clusters of microglia enriched for biological pathways implicating defined myeloid roles. We detected several previously unrecognized microglia populations in human AD brain, including three internalization and trafficking subtypes that were heterogeneous in their metabolic and inflammatory signatures. One of these endolysosomal subtypes is larger in AD individuals and was uniquely enriched for genes involved in nucleic acid detection and activation of interferon signaling. This inflammatory endolysosomal cluster also differentially regulated expression of genes associated with AD risk by genome wide association studies. We also identified a cluster of microglia with upregulated cell cycle and DNA repair genes that is proportionately larger in control individuals. Within cluster comparisons demonstrate that in AD brain, homeostatic microglia subpopulations upregulate inflammatory gene expression. These results highlight the heterogenous nature of the microglia response to AD pathology and will inform efforts to target specific subtypes of microglia in the development of novel AD therapies.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a sub-threshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked up-regulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular co-localization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation, that may be involved in IPC-mediated protection.

Objective Autosomal‐dominant familial Alzheimer disease (AD) is caused by by variants in presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP). Previously, we reported a rare PSEN2 frameshift variant in an early‐onset AD case (PSEN2 p.K115Efs*11). In this study, we characterize a second family with the same variant and analyze cellular transcripts from both patient fibroblasts and brain lysates. Methods We combined genomic, neuropathological, clinical, and molecular techniques to characterize the PSEN2 K115Efs*11 variant in two families. Results Neuropathological and clinical evaluation confirmed the AD diagnosis in two individuals carrying the PSEN2 K115Efs*11 variant. A truncated transcript from the variant allele is detectable in patient fibroblasts while levels of wild‐type PSEN2 transcript and protein are reduced compared to controls. Functional studies to assess biological consequences of the variant demonstrated that PSEN2 K115Efs*11 fibroblasts secrete less Aβ 1–40 compared to controls, indicating abnormal γ‐secretase activity. Analysis of PSEN2 transcript levels in brain tissue revealed alternatively spliced PSEN2 products in patient brain as well as in sporadic AD and age‐matched control brain. Interpretation These data suggest that PSEN2 K115Efs*11 is a likely pathogenic variant associated with AD. We uncovered novel PSEN2 alternative transcripts in addition to previously reported PSEN2 splice isoforms associated with sporadic AD. In the context of a frameshift, these alternative transcripts return to the canonical reading frame with potential to generate deleterious protein products. Our findings suggest novel potential mechanisms by which PSEN variants may influence AD pathogenesis, highlighting the complexity underlying genetic contribution to disease risk.

Immunotherapy for hematologic malignancies with CD19-directed CAR T cells is associated with neurotoxicity in about 40% of patients. Systemic cytokine release syndrome, endothelial activation, and disruption of endothelial integrity have all been associated with neurotoxicity, but it remains unclear how these mechanisms interact and how they lead to neurologic dysfunction. We developed a syngeneic mouse model which manifests systemic cytokine release and behavioral abnormalities within 3-5 days after infusion of high-dose murine CD19-CAR T cells. Histologic examination revealed widespread brain hemorrhages, diffuse extravascular IgG deposition, loss of capillary pericyte coverage and increased prevalence of string capillaries. In vivo two-photon imaging of blood flow revealed plugging of >10% of capillaries by leukocytes, associated with regions of localized hypoxia. These data reveal capillary obstruction and associated brain hypoxia and microvascular decline as a potential basis for neurotoxicity during CD19-CAR T cell treatment in humans, which may be amenable to therapeutic interventions.

Background Computational phenotypes are most often combinations of patient billing codes that are highly predictive of disease using electronic health records (EHR). In the case of rare diseases that can only be diagnosed by genetic testing, computational phenotypes identify patient cohorts for genetic testing and possible diagnosis. This article details the validation of a computational phenotype for PTEN hamartoma tumor syndrome (PHTS) against the EHR of patients at three collaborating clinical research centers: Boston Children's Hospital, Children's National Hospital, and the University of Washington. Methods A combination of billing codes from the International Classification of Diseases versions 9 and 10 (ICD-9 and ICD-10) for diagnostic criteria postulated by a research team at Cleveland Clinic was used to identify patient cohorts for genetic testing from the clinical data warehouses at the three research centers. Subsequently, the EHR—including billing codes, clinical notes, and genetic reports—of these patients were reviewed by clinical experts to identify patients with PHTS. Results The PTEN genetic testing yield of the computational phenotype, the number of patients who needed to be genetically tested for incidence of pathogenic PTEN gene variants, ranged from 82 to 94% at the three centers. Conclusions Computational phenotypes have the potential to enable the timely and accurate diagnosis of rare genetic diseases such as PHTS by identifying patient cohorts for genetic sequencing and testing.

Microglia are the innate immune cells of the central nervous system that adopt rapid functional changes in response to Damage Associated Molecular Patterns, including aggregated β-Amyloid (Aβ) found in Alzheimer's disease (AD). microRNAs (miRNAs) are post-transcriptional modulators that influence the timing and magnitude of microglia inflammatory responses by downregulating the expression of inflammatory effectors. Recent studies implicate miR-155, a miRNA known to regulate inflammatory responses, in the pathogenesis of neurodegenerative disorders including multiple sclerosis, ALS, familial Parkinson's disease, and AD. In this work, we asked if miR-155 expression in microglia modifies cellular behaviors in response to fibrillar Aβ(1-42) (fAβ(1-42)), in vitro. We hypothesized that in microglia, miR-155 expression would impact the internalization and catabolism of extracellular fAβ(1-42). Primary microglia stimulated with lipopolysaccharide demonstrate fast upregulation of miR-155 followed by delayed upregulation of miR-146a, an anti-inflammatory miRNA. Conditional overexpression of miR-155 in microglia resulted in significant upregulation of miR-146a. Conditional deletion of miR-155 promoted transit of fAβ(1-42) to low-pH compartments where catabolism occurs, while miR-155 overexpression decreases fAβ(1-42) catabolism. Uptake of fAβ(1-42) across the plasma membrane increased with both up and downregulation of miR-155 expression. Taken together, our results support the hypothesis that inflammatory signaling influences the ability of microglia to catabolize fAβ(1-42) through interconnected mechanisms modulated by miR-155. Understanding how miRNAs modulate the ability of microglia to catabolize fAβ(1-42) will further elucidate the role of cellular players and molecular crosstalk in AD pathophysiology.

Chimeric antigen receptor (CAR) T cells provide new therapeutic options for patients with relapsed/refractory hematologic malignancies. However, neurotoxicity is a frequent, and potentially fatal, complication. The spectrum of manifestations ranges from delirium and language dysfunction to seizures, coma, and fatal cerebral edema. This novel syndrome has been designated immune effector cell–associated neurotoxicity syndrome (ICANS). In this review, we draw an arc from our current understanding of how systemic and potentially local cytokine release act on the CNS, toward possible preventive and therapeutic approaches. We systematically review reported correlations of secreted inflammatory mediators in the serum/plasma and cerebrospinal fluid with the risk of ICANS in patients receiving CAR T cell therapy. Possible pathophysiologic impacts on the CNS are covered in detail for the most promising candidate cytokines, including IL-1, IL-6, IL-15, and GM-CSF. To provide insight into possible final common pathways of CNS inflammation, we place ICANS into the context of other systemic inflammatory conditions that are associated with neurologic dysfunction, including sepsis-associated encephalopathy, cerebral malaria, thrombotic microangiopathy, CNS infections, and hepatic encephalopathy. We then review in detail what is known about systemic cytokine interaction with components of the neurovascular unit, including endothelial cells, pericytes, and astrocytes, and how microglia and neurons respond to systemic inflammatory challenges. Current therapeutic approaches, including corticosteroids and blockade of IL-1 and IL-6 signaling, are reviewed in the context of what is known about the role of cytokines in ICANS. Throughout, we point out gaps in knowledge and possible new approaches for the investigation of the mechanism, prevention, and treatment of ICANS.

The temporal molecular changes that lead to disease onset and progression in Alzheimer’s disease (AD) are still unknown. Here we develop a temporal model for these unobserved molecular changes with a manifold learning method applied to RNA-Seq data collected from human postmortem brain samples collected within the ROS/MAP and Mayo Clinic RNA-Seq studies. We define an ordering across samples based on their similarity in gene expression and use this ordering to estimate the molecular disease stage–or disease pseudotime-for each sample. Disease pseudotime is strongly concordant with the burden of tau (Braak score, P = 1.0 × 10−5), Aβ (CERAD score, P = 1.8 × 10−5), and cognitive diagnosis (P = 3.5 × 10−7) of late-onset (LO) AD. Early stage disease pseudotime samples are enriched for controls and show changes in basic cellular functions. Late stage disease pseudotime samples are enriched for late stage AD cases and show changes in neuroinflammation and amyloid pathologic processes. We also identify a set of late stage pseudotime samples that are controls and show changes in genes enriched for protein trafficking, splicing, regulation of apoptosis, and prevention of amyloid cleavage pathways. In summary, we present a method for ordering patients along a trajectory of LOAD disease progression from brain transcriptomic data., The limited understanding of the temporal molecular changes in late-onset Alzheimer’s disease hinder the development of therapeutic treatment. The authors use manifold learning to develop a molecular model for disease progression from RNASeq data from human postmortem brain samples.

Repeated cycles of alcohol intoxication and withdrawal both induce profound changes in gene expression that can contribute to the physiological and behavioral consequences of ethanol. Since neuroinflammation is an important consequence of these changes, we used a novel strategy to investigate the impact of repeated cycles of chronic intermittent ethanol vapor and withdrawal on the RNAs actively undergoing translation in striatal microglia. RiboTag was selectively expressed in the microglia of transgenic mice and was used to immunopurify the RNA “translatome” from striatal microglia, yielding a snapshot of RNA translation during alcohol intoxication and after 8 hours of withdrawal. We obtained highly enriched microglial RNAs and analyzed these in individual animals by deep sequencing. We found a dramatic shift in gene expression during acute intoxication compared to air-exposed controls, with increases in genes and pathways associated with cytokine signaling, indicating increased neuroinflammation and microglial activation. After 8 hours of ethanol withdrawal, many inflammatory pathways remained upregulated but phagocytotic and proapoptotic pathways were increased. Using an unbiased bioinformatic method, weighted gene coexpression network analysis, multiple differentially expressed gene modules were identified. One in particular was differentially expressed in ethanol intoxicated vs. withdrawing animals, and there was a strong correlation between the centrality of the genes to this gene network and their individual statistical significance in differential expression. The unfolded protein response was over-represented in this network after withdrawal. The induction of this pathway in microglia is important since this cellular stress response can either lead towards restoration of normal function or apoptosis.

Microglia have recently been implicated in dependence to opioids. To investigate this directly, we used RNA sequencing of ribosome associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and then the precipitation of withdrawal by naloxone. We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal, and bioinformatics analysis revealed an intriguing upregulation of cAMP-associated genes that are involved in microglial motility, morphology, and interactions with neurons. Three-dimensional histological reconstruction of microglia revealed changes in process branching and termination following opioid tolerance that were rapidly reversed during withdrawal and were consistent with cAMP effects on microglia morphology. Direct activation of Gi-coupled DREADD receptors in microglia, rather than mimicking the effects of morphine, exacerbated opioid withdrawal. Together these indicate that microglial response to cAMP signaling can mitigate the rapid manifestations of opioid withdrawal.

Histone deacetylases (HDACs) catalyze acetyl group removal from histone proteins, leading to altered chromatin structure and gene expression. HDAC2 is highly expressed in adult brain, and HDAC2 levels are elevated in Alzheimer's disease (AD) brain. We previously reported that neuron-specific splice isoforms of Endophilin-B1 (Endo-B1) promote neuronal survival, but are reduced in human AD brain and mouse models of AD and stroke. Here, we demonstrate that HDAC2 suppresses Endo-B1 expression. HDAC2 knockdown or knockout enhances expression of Endo-B1. Conversely, HDAC2 overexpression decreases Endo-B1 expression. We also demonstrate that neurons exposed to beta-amyloid increase HDAC2 and reduce histone H3 acetylation while HDAC2 knockdown prevents Aβ induced loss of histone H3 acetylation, mitochondrial dysfunction, caspase-3 activation, and neuronal death. The protective effect of HDAC2 knockdown was abrogated by Endo-B1 shRNA and in Endo-B1-null neurons, suggesting that HDAC2-induced neurotoxicity is mediated through suppression of Endo-B1. HDAC2 overexpression also modulates neuronal expression of mitofusin2 (Mfn2) and mitochondrial fission factor (MFF), recapitulating the pattern of change observed in AD. HDAC2 knockout mice demonstrate reduced injury in the middle cerebral artery occlusion with reperfusion (MCAO/R) model of cerebral ischemia demonstrating enhanced neuronal survival, minimized loss of Endo-B1, and normalized expression of Mfn2. These findings support the hypothesis that HDAC2 represses Endo-B1, sensitizing neurons to mitochondrial dysfunction and cell death in stroke and AD.

The temporal molecular changes that lead to disease onset and progression in Alzheimer's disease (AD) are still unknown. Here we develop a temporal model for these unobserved molecular changes with a manifold learning method applied to RNA-Seq data collected from human postmortem brain samples collected within the ROS/MAP and Mayo Clinic RNA-Seq studies. We define an ordering across samples based on their similarity in gene expression and use this ordering to estimate the molecular disease stage-or disease pseudotime-for each sample. Disease pseudotime is strongly concordant with the burden of tau (Braak score, P = 1.0 × 10

OBJECTIVE Immunotherapy for hematologic malignancies with CD19-directed CAR T cells is complicated by neurotoxicity in approximately 40% of patients. We have previously reported evidence of glial injury in pediatric patients with CAR T neurotoxicity by elevated CSF levels of GFAP and S100b. We now hypothesize that NFL is also a useful biomarker of neuronal injury related to abnormal blood-brain-barrier and glial function. METHODS We used the Mesoscale Discovery platform to measure CSF and serum NFL levels in a consecutive cohort of 43 pediatric patients with B cell ALL who received CD19-directed CAR T cells. In addition, we will present expansion cohort measurements of NFL and GFAP (N=95). RESULTS CSF NFL levels prior to CAR T cell infusion positively correlated with the risk of subsequently developing severe neurotoxicity (no neurotoxicity, median 275pg/mL, mild 378pg/mL, severe 951pg/mL, P=0.0182 for severe vs none, P=0.0458 for severe vs mild). During neurotoxicity, mean CSF NFL levels increased to 1179pg/mL (mild neurotoxicity, P=0.0338) and 1345 pg/mL (severe neurotoxicity, P=0.0148), respectively. In serum, pretreatment NFL levels were highly abnormal in many patients (median 368pg/mL, range 10–56,321pg/mL; healthy control median 4pg/mL, range 1–7.5pg/mL). However, there was no correlation with neurotoxicity, history of CNS radiation, peripheral neuropathy, stem cell transplant, or number of prior chemotherapies. Day 7 serum NFL levels did not change significantly (median 439pg/mL, range 5–17,439pg/mL, P=0.3254). CONCLUSION We conclude that CSF NFL is promising biomarker of CAR T neurotoxicity risk and severity. The abnormal baseline serum NFL concentrations remain unexplained and require further study.

There is a growing understanding that aberrant GLIA function is an underlying factor in psychiatric and neurological disorders. As drug discovery efforts begin to focus on glia-related targets, a key gap in knowledge includes the availability of validated biomarkers to help determine which patients suffer from dysfunction of glial cells or who may best respond by targeting glia-related drug mechanisms. Biomarkers are biological variables with a significant relationship to parameters of disease states and can be used as surrogate markers of disease pathology, progression, and/or responses to drug treatment. For example, imaging studies of the CNS enable localization and characterization of anatomical lesions without the need to isolate tissue for biopsy. Many biomarkers of disease pathology in the CNS involve assays of glial cell function and/or response to injury. Each major glia subtype (oligodendroglia, astroglia and microglia) are connected to a number of important and useful biomarkers. Here, we describe current and emerging glial based biomarker approaches for acute CNS injury and the major categories of chronic nervous system dysfunction including neurodegenerative, neuropsychiatric, neoplastic, and autoimmune disorders of the CNS. These descriptions are highlighted in the context of how biomarkers are employed to better understand the role of glia in human CNS disease and in the development of novel therapeutic treatments. GLIA 2016;64:1755-1771.

Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

© 2015 The New York Academy of Sciences. An emerging aspect of neuronal-glial interactions is the connection glial cells have to synapses. Mounting research now suggests a far more intimate relationship than previously recognized. Moreover, the current evidence implicating synapse loss in neurodegenerative disease etiology is overwhelming, but the role of glia in the process of synaptic degeneration has only recently been considered in earnest. Each main class of glial cell, including astrocytes, oligodendrocytes, and microglia, performs crucial and multifaceted roles in the maintenance of synaptic function and excitability. As such, aging and/or neuronal stress from disease-related misfolded proteins may involve disruption of multiple non-cell-autonomous synaptic support systems that are mediated by neighboring glia. In addition, glial cell activation induced by injury, ischemia, or neurodegeneration is thought to greatly alter the behavior of glial cells toward neuronal synapses, suggesting that neuroinflammation potentially contributes to synapse loss primarily mediated by altered glial functions. This review discusses recent evidence highlighting novel roles for glial cells at neuronal synapses and in the maintenance of neuronal connectivity, focusing primarily on their implications for neurodegenerative disease research.

SummaryChronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a−/− mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155fl/fl mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.

The innate immune system has been implicated in several neurodegenerative diseases, including HIV-1–associated dementia. In this study, we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-using viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To characterize further the neuroprotective effect of CCR5 deficiency we performed a genome-wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and nontransgenic controls. A comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly upregulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion, whereas inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-using gp120. However, the combination of pharmacologic CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-using gp120, thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Our study provides evidence for an indirect pathologic role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury.

Neuroinflammation occurs in acute and chronic CNS injury, including stroke, traumatic brain injury, and neurodegenerative diseases. Microglia are specialized resident myeloid cells that mediate CNS innate immune responses. Disease-relevant stimuli, such as reactive oxygen species (ROS), can influence microglia activation. Previously, we observed that p53, a ROS-responsive transcription factor, modulates microglia behaviors in vitro and in vivo, promoting proinflammatory functions and suppressing downregulation of the inflammatory response and tissue repair. In this article we describe a novel mechanism by which p53 modulates the functional differentiation of microglia both in vitro and in vivo. Adult microglia from p53-deficient mice have increased expression of the anti-inflammatory transcription factor c-Maf. To determine how p53 negatively regulates c-Maf, we examined the impact of p53 on known c-Maf regulators. MiR-155 is a microRNA that targets c-Maf. We observed that cytokine-induced expression of miR-155 was suppressed in p53-deficient microglia. Furthermore, Twist2, a transcriptional activator of c-Maf, is increased in p53-deficient microglia. We identified recognition sites in the 3′ untranslated region of Twist2 mRNA that are predicted to interact with two p53-dependent microRNAs: miR-34a and miR-145. In this article, we demonstrate that miR-34a and -145 are regulated by p53 and negatively regulate Twist2 and c-Maf expression in microglia and the RAW macrophage cell line. Taken together, these findings support the hypothesis that p53 activation induced by local ROS or accumulated DNA damage influences microglia functions and that one specific molecular target of p53 in microglia is c-Maf.

We describe the development of fetal brain lesions after Zika virus (ZIKV) inoculation in a pregnant pigtail macaque. Periventricular lesions developed within 10 d and evolved asymmetrically in the occipital-parietal lobes. Fetal autopsy revealed ZIKV in the brain and significant cerebral white matter hypoplasia, periventricular white matter gliosis, and axonal and ependymal injury. Our observation of ZIKV-associated fetal brain lesions in a nonhuman primate provides a model for therapeutic evaluation.

Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794.

The tumor-suppressor protein p53 belongs to a family of proteins that play pivotal roles in multiple cellular functions including cell proliferation, cell death, genome stability, and regulation of inflammation. Neuroinflammation is a common feature of central nervous system (CNS) pathology, and microglia are the specialized resident population of CNS myeloid cells that initiate innate immune responses. Microglia maintain CNS homeostasis through pathogen containment, phagocytosis of debris, and initiation of tissue-repair cascades. However, an unregulated pro-inflammatory response can lead to tissue injury and dysfunction in both acute and chronic inflammatory states. Therefore, regulation of the molecular signals that control the induction, magnitude, and resolution of inflammation are necessary for optimal CNS health. We and others have described a novel mechanism by which p53 transcriptional activity modulates microglia behaviors in vitro and in vivo. Activation of p53 induces expression of microRNAs (miRNAs) that support microglia pro-inflammatory functions and suppress anti-inflammatory and tissue repair behaviors. In this review, we introduce the previously described roles of the p53 signaling network and discuss novel functions of p53 in the microglia-mediated inflammatory response in CNS health and disease. Ultimately, improved understanding of the molecular regulators modulated by p53 transcriptional activity in microglia will enhance the development of rational therapeutic strategies to harness the homeostatic and tissue repair functions of microglia.

Spinocerebellar ataxia type 7 (SCA7) is a dominantly inherited neurodegenerative disorder caused by a CAG - polyglutamine (polyQ) repeat expansion in the ataxin-7 gene. In polyQ disorders, synaptic dysfunction and neurodegeneration may develop prior to symptom onset. However, conditional expression studies of polyQ disease models demonstrate that suppression of gene expression can yield complete reversal of established behavioral abnormalities. To determine if SCA7 neurological and neurodegenerative phenotypes are reversible, we crossed PrP-floxed-SCA7-92Q BAC transgenic mice with a tamoxifen-inducible Cre recombinase transgenic line, CAGGS-Cre-ER™. PrP-floxed-SCA7-92Q BAC;CAGGS-Cre-ER™ bigenic mice were treated with a single dose of tamoxifen 1 month after the onset of detectable ataxia, which resulted in ~50% reduction of polyQ-ataxin-7 expression. Tamoxifen treatment halted or reversed SCA7 motor symptoms, reduced ataxin-7 aggregation in Purkinje cells (PCs), and prevented loss of climbing fiber (CF)-PC synapses in comparison to vehicle-treated bigenic animals and tamoxifen-treated PrP-floxed-SCA7-92Q BAC single transgenic mice. Despite this phenotype rescue, reduced ataxin-7 expression did not result in full recovery of cerebellar molecular layer thickness or prevent Bergmann glia degeneration. These results demonstrate that suppression of mutant gene expression by only 50% in a polyQ disease model can have a significant impact on disease phenotypes, even when initiated after the onset of detectable behavioral deficits. The findings reported here are consistent with the emerging view that therapies aimed at reducing neurotoxic gene expression hold the potential to halt or reverse disease progression in afflicted patients, even after the onset of neurological disability.

Neurodegenerative diseases have been intensively studied, but a comprehensive understanding of their pathogenesis remains elusive. An increasing body of evidence suggests that non-cell autonomous processes play critical roles during the initiation, and spatiotemporal progression or propagation of the dominant pathology. Here we review findings highlighting the importance of pathological cell-cell communication in neurodegenerative disease. We focus primarily on the accumulating evidence suggesting dysfunctional cross-talk between neurons and astroglia, neurons and innate immune system cells, as well as cellular processes leading to transmission of pathogenic proteins between cells. Insights into the complex intercellular perturbations underlying neurodegeneration will enhance our efforts to develop effective therapeutic approaches for preventing or reversing symptomatic progression in this devastating class of human diseases.

Spinocerebellar ataxia type 7 (SCA7) is a dominantly inherited disorder characterized by cerebellum and brainstem neurodegeneration. SCA7 is caused by a CAG/polyglutamine (polyQ) repeat expansion in the ataxin-7 gene. We previously reported that directed expression of polyQ-ataxin-7 in Bergmann glia (BG) in transgenic mice leads to ataxia and non-cell-autonomous Purkinje cell (PC) degeneration. To further define the cellular basis of SCA7, we derived a conditional inactivation mouse model by inserting a loxP-flanked ataxin-7 cDNA with 92 repeats into the translational start site of the murine prion protein (PrP) gene in a bacterial artificial chromosome (BAC). ThePrP-floxed-SCA7-92Q BACmice developed neurological disease, and exhibited cerebellar degeneration and BG process loss. To inactivate polyQ-ataxin-7 expression in specific cerebellar cell types, we crossedPrP-floxed-SCA7-92Q BACmice withGfa2-Cretransgenic mice (to direct Cre to BG) orPcp2-Cretransgenic mice (which yields Cre in PCs and inferior olive). Excision of ataxin-7 from BG partially rescued the behavioral phenotype, but did not prevent BG process loss or molecular layer thinning, while excision of ataxin-7 from PCs and inferior olive provided significantly greater rescue and prevented both pathological changes, revealing a non-cell-autonomous basis for BG pathology. When we prevented expression of mutant ataxin-7 in BG, PCs, and inferior olive by derivingGfa2-Cre;Pcp2-Cre;PrP-floxed-SCA7-92Q BACtriple transgenic mice, we noted a dramatic improvement in SCA7 disease phenotypes. These findings indicate that SCA7 disease pathogenesis involves a convergence of alterations in a variety of different cell types to fully recapitulate the cerebellar degeneration.

Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia have proinflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV-associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here, we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete proinflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis, and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a proinflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. © 2011 Wiley-Liss, Inc.

BACKGROUND AND PURPOSE: Surface disruption, either ulceration or fibrous cap rupture, has been identified as a key feature of the unstable atherosclerotic plaque. In this prospective observational study, we sought to determine the characteristics of the carotid lesion that predict the development of new surface disruption. MATERIALS AND METHODS: One hundred eight asymptomatic individuals with 50%–79% carotid stenosis underwent carotid MR imaging at baseline and at 3 years. Multicontrast imaging criteria were used to determine the presence or absence of calcification, LRNC, intraplaque hemorrhage, and surface disruption. Volume measurements of plaque morphology and the LRNC and calcification, when present, were collected. RESULTS: At baseline, 21.3% (23/108) of participants were identified with a surface disruption. After 3 years, 9 (10.6%) of the remaining 85 individuals without disruption at baseline developed a new surface disruption during follow-up. Among all baseline variables associated with new surface disruption during regression analysis, the proportion of wall volume occupied by the LRNC (percentage LRNC volume; OR per 5% increase, 2.6; 95% CI, 1.5–4.6) was the strongest classifier (AUC = 0.95) during ROC analysis. New surface disruption was associated with a significant increase in percentage LRNC volume (1.7 ± 2.0% per year, P = .035). CONCLUSIONS: This prospective investigation of asymptomatic individuals with 50%–79% stenosis provides compelling evidence that LRNC size may govern the risk of future surface disruption. Identification of carotid plaques in danger of developing new surface disruption may prove clinically valuable for preventing the transition from stable to unstable atherosclerotic disease.

The human immunodeficiency virus (HIV) invades the central nervous system early in the course of infection and establishes a protected viral reservoir. However, neurocognitive consequences of HIV infection, known collectively as HIV-associated neurocognitive disorders (HAND), develop in only a small portion of infected patients. The precise mechanisms of pathogenesis involved in HIV-induced central nervous system injury are still not completely understood. In particular, most theories of HAND pathogenesis cannot account for either the selective vulnerability of specific neuronal populations to HIV-induced neurodegeneration or why only a subset of patients develop clinically detectable nervous system disease. Epidemiological and virological studies have identified a variety of host and viral factors that are associated with increased risk of developing HAND. Some host factors that predispose HIV-infected patients to HAND overlap with those associated with Alzheimer’s disease (AD), suggesting the possibility that common pathogenic mechanisms may participate in both diseases. Here, we will review reports of host and viral factors associated with HAND and place these studies in the context of the data employed to support current theories regarding the molecular and cellular mechanisms that lead to HIV-induced neurodegeneration with additional focus on mechanisms common to AD pathogenesis.