Ali Marandi Ghoddousi, R;Magalong, VM;Kamitakahara, AK;Levitt, P;
PMID: 36313803 | DOI: 10.1016/j.crmeth.2022.100316
Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.
Wang, Y;Eddison, M;Fleishman, G;Weigert, M;Xu, S;Wang, T;Rokicki, K;Goina, C;Henry, FE;Lemire, AL;Schmidt, U;Yang, H;Svoboda, K;Myers, EW;Saalfeld, S;Korff, W;Sternson, SM;Tillberg, PW;
PMID: 34875226 | DOI: 10.1016/j.cell.2021.11.024
Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.
Zhang, X;Liu, Y;Hong, X;Li, X;Meshul, CK;Moore, C;Yang, Y;Han, Y;Li, WG;Qi, X;Lou, H;Duan, S;Xu, TL;Tong, X;
PMID: 34593806 | DOI: 10.1038/s41467-021-25956-y
NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress.
Maksymetz, J;Byun, NE;Luessen, DJ;Li, B;Barry, RL;Gore, JC;Niswender, CM;Lindsley, CW;Joffe, ME;Conn, PJ;
PMID: 34731619 | DOI: 10.1016/j.celrep.2021.109950
Evidence for prefrontal cortical (PFC) GABAergic dysfunction is one of the most consistent findings in schizophrenia and may contribute to cognitive deficits. Recent studies suggest that the mGlu1 subtype of metabotropic glutamate receptor regulates cortical inhibition; however, understanding the mechanisms through which mGlu1 positive allosteric modulators (PAMs) regulate PFC microcircuit function and cognition is essential for advancing these potential therapeutics toward the clinic. We report a series of electrophysiology, optogenetic, pharmacological magnetic resonance imaging, and animal behavior studies demonstrating that activation of mGlu1 receptors increases inhibitory transmission in the prelimbic PFC by selective excitation of somatostatin-expressing interneurons (SST-INs). An mGlu1 PAM reverses cortical hyperactivity and concomitant cognitive deficits induced by N-methyl-d-aspartate (NMDA) receptor antagonists. Using in vivo optogenetics, we show that prelimbic SST-INs are necessary for mGlu1 PAM efficacy. Collectively, these findings suggest that mGlu1 PAMs could reverse cortical GABAergic deficits and exhibit efficacy in treating cognitive dysfunction in schizophrenia.
Inhibition of the cGAS-STING pathway ameliorates the premature senescence hallmarks of Ataxia-Telangiectasia brain organoids
Aguado, J;Chaggar, HK;Gómez-Inclán, C;Shaker, MR;Leeson, HC;Mackay-Sim, A;Wolvetang, EJ;
PMID: 34459078 | DOI: 10.1111/acel.13468
Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.
Spatially patterned excitatory neuron subtypes and projections of the claustrum
Erwin, SR;Bristow, BN;Sullivan, KE;Kendrick, RM;Marriott, B;Wang, L;Clements, J;Lemire, AL;Jackson, J;Cembrowski, MS;
PMID: 34397382 | DOI: 10.7554/eLife.68967
The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.
Atlan G, Terem A, Peretz-Rivlin N, Sehrawat K, Gonzales BJ, Pozner G, Tasaka G, Goll Y, Refaeli R, Zviran O, Lim BK, Groysman M, Goshen I, Mizrahi A, Nelken I, Citri A.
PMID: 30122531 | DOI: 10.1016/j.cub.2018.06.068
A barrage of information constantly assaults our senses, of which only a fraction is relevant at any given point in time. However, the neural circuitry supporting the suppression of irrelevant sensory distractors is not completely understood. The claustrum, a circuit hub with vast cortical connectivity, is an intriguing brain structure, whose restrictive anatomy, thin and elongated, has precluded functional investigation. Here, we describe the use of Egr2-CRE mice to access genetically defined claustral neurons. Utilizing conditional viruses for anterograde axonal labeling and retrograde trans-synaptic tracing, we validated this transgenic model for accessing the claustrum and extended the known repertoire of claustral input/output connectivity. Addressing the function of the claustrum, we inactivated CLEgr2+ neurons, chronically as well as acutely, in mice performing an automated two-alternative forced-choice behavioral task. Strikingly, inhibition of CLEgr2+ neurons did not significantly impact task performance under varying delay times and cue durations, but revealed a selective role for the claustrum in supporting performance in the presence of an irrelevant auditory distractor. Further investigation of behavior, in the naturalistic maternal pup-retrieval task, replicated the result of sensitization to an auditory distractor following inhibition of CLEgr2+ neurons. Initiating investigation into the underlying mechanism, we found that activation of CLEgr2+ neurons modulated cortical sensory processing, suppressing tone representation in the auditory cortex. This functional study, utilizing selective genetic access, implicates the claustrum in supporting resilienceto distraction, a fundamental aspect of attention.
Shukla R, Prevot TD, French L, Isserlin R, Rocco BR, Banasr M, Bader GD, Sibille E.
PMID: - | DOI: 10.1016/j.celrep.2018.09.034
Background Aging is accompanied by altered thinking (cognition) and feeling (mood), functions that depend on information processing by brain cortical cell microcircuits. We hypothesized that age-associated long-term functional and biological changes are mediated by gene transcriptomic changes within neuronal cell-types forming cortical microcircuits, namely excitatory pyramidal cells (PYC) and inhibitory GABAergic neurons expressing vasoactive intestinal peptide (Vip), somatostatin (Sst) and parvalbumin (Pvalb). Methods To test this hypothesis, we assessed locomotor, anxiety-like and cognitive behavioral changes between young (2 months, n=9) and old (22 months, n=12) male C57BL/6 mice, and performed frontal cortex cell-type specific molecular profiling, using laser-capture microscopy and RNA sequencing. Results were analyzed by neuroinformatics and validated by fluorescent in situ hybridization. Results Old-mice displayed increased anxiety and reduced working memory. The four cell-types displayed distinct age-related transcriptomes and biological pathway profiles, affecting metabolic and cell signaling pathways, and selective markers of neuronal vulnerability (Ryr3), resilience (Oxr1), and mitochondrial dynamics (Opa1), suggesting high age-related vulnerability of PYCs, and variable degree of adaptation in GABAergic neurons. Correlations between gene expression and behaviors suggest that changes in cognition and anxiety associated with age are partly mediated by normal age-related cell changes, and that additional age-independent decreases in synaptic and signaling pathways, notably in PYC and SST-neurons further contribute to behavioral changes. Conclusions Our study demonstrates cell-dependent differential vulnerability and coordinated cell-specific cortical microcircuit molecular changes with age. Collectively, the results suggest intrinsic molecular links between aging, cognition and mood-related behaviors with SST-neurons contributing evenly to both behavioral conditions.
Burda, JE;O'Shea, TM;Ao, Y;Suresh, KB;Wang, S;Bernstein, AM;Chandra, A;Deverasetty, S;Kawaguchi, R;Kim, JH;McCallum, S;Rogers, A;Wahane, S;Sofroniew, MV;
PMID: 35614216 | DOI: 10.1038/s41586-022-04739-5
Astrocytes respond to injury and disease in the central nervous system with reactive changes that influence the outcome of the disorder1-4. These changes include differentially expressed genes (DEGs) whose contextual diversity and regulation are poorly understood. Here we combined biological and informatic analyses, including RNA sequencing, protein detection, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and conditional gene deletion, to predict transcriptional regulators that differentially control more than 12,000 DEGs that are potentially associated with astrocyte reactivity across diverse central nervous system disorders in mice and humans. DEGs associated with astrocyte reactivity exhibited pronounced heterogeneity across disorders. Transcriptional regulators also exhibited disorder-specific differences, but a core group of 61 transcriptional regulators was identified as common across multiple disorders in both species. We show experimentally that DEG diversity is determined by combinatorial, context-specific interactions between transcriptional regulators. Notably, the same reactivity transcriptional regulators can regulate markedly different DEG cohorts in different disorders; changes in the access of transcriptional regulators to DNA-binding motifs differ markedly across disorders; and DEG changes can crucially require multiple reactivity transcriptional regulators. We show that, by modulating reactivity, transcriptional regulators can substantially alter disorder outcome, implicating them as therapeutic targets. We provide searchable resources of disorder-related reactive astrocyte DEGs and their predicted transcriptional regulators. Our findings show that transcriptional changes associated with astrocyte reactivity are highly heterogeneous and are customized from vast numbers of potential DEGs through context-specific combinatorial transcriptional-regulator interactions.
bioRxiv : the preprint server for biology
Toro, CA;Johnson, K;Hansen, J;Siddiq, MM;Vásquez, W;Zhao, W;Graham, ZA;Sáez, JC;Iyengar, R;Cardozo, CP;
PMID: 36824813 | DOI: 10.1101/2023.02.15.528337
Membrane channels such as connexins (Cx), pannexins (Panx) and P2X 7 receptors (P2X 7 R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X 7 R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.
Brain : a journal of neurology
Lee, MH;Perl, DP;Steiner, J;Pasternack, N;Li, W;Maric, D;Safavi, F;Horkayne-Szakaly, I;Jones, R;Stram, MN;Moncur, JT;Hefti, M;Folkerth, RD;Nath, A;
PMID: 35788639 | DOI: 10.1093/brain/awac151
The underlying mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to acute and long-term neurological manifestations remains obscure. We aimed to characterize the neuropathological changes in patients with coronavirus disease 2019 and determine the underlying pathophysiological mechanisms. In this autopsy study of the brain, we characterized the vascular pathology, the neuroinflammatory changes and cellular and humoral immune responses by immunohistochemistry. All patients died during the first wave of the pandemic from March to July 2020. All patients were adults who died after a short duration of the infection, some had died suddenly with minimal respiratory involvement. Infection with SARS-CoV-2 was confirmed on ante-mortem or post-mortem testing. Descriptive analysis of the pathological changes and quantitative analyses of the infiltrates and vascular changes were performed. All patients had multifocal vascular damage as determined by leakage of serum proteins into the brain parenchyma. This was accompanied by widespread endothelial cell activation. Platelet aggregates and microthrombi were found adherent to the endothelial cells along vascular lumina. Immune complexes with activation of the classical complement pathway were found on the endothelial cells and platelets. Perivascular infiltrates consisted of predominantly macrophages and some CD8+ T cells. Only rare CD4+ T cells and CD20+ B cells were present. Astrogliosis was also prominent in the perivascular regions. Microglial nodules were predominant in the hindbrain, which were associated with focal neuronal loss and neuronophagia. Antibody-mediated cytotoxicity directed against the endothelial cells is the most likely initiating event that leads to vascular leakage, platelet aggregation, neuroinflammation and neuronal injury. Therapeutic modalities directed against immune complexes should be considered.
Lamoureux, L;Sajesh, B;Slota, JA;Medina, SJ;Mayor, M;Frost, KL;Warner, B;Manguiat, K;Wood, H;Kobasa, D;Booth, SA;
PMID: 35746689 | DOI: 10.3390/v14061218
The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement.
