Jiwaji, Z;Tiwari, SS;Avilés-Reyes, RX;Hooley, M;Hampton, D;Torvell, M;Johnson, DA;McQueen, J;Baxter, P;Sabari-Sankar, K;Qiu, J;He, X;Fowler, J;Febery, J;Gregory, J;Rose, J;Tulloch, J;Loan, J;Story, D;McDade, K;Smith, AM;Greer, P;Ball, M;Kind, PC;Matthews, PM;Smith, C;Dando, O;Spires-Jones, TL;Johnson, JA;Chandran, S;Hardingham, GE;
PMID: 35013236 | DOI: 10.1038/s41467-021-27702-w
Alzheimer's disease (AD) alters astrocytes, but the effect of Aß and Tau pathology is poorly understood. TRAP-seq translatome analysis of astrocytes in APP/PS1 ß-amyloidopathy and MAPTP301S tauopathy mice revealed that only Aß influenced expression of AD risk genes, but both pathologies precociously induced age-dependent changes, and had distinct but overlapping signatures found in human post-mortem AD astrocytes. Both Aß and Tau pathology induced an astrocyte signature involving repression of bioenergetic and translation machinery, and induction of inflammation pathways plus protein degradation/proteostasis genes, the latter enriched in targets of inflammatory mediator Spi1 and stress-activated cytoprotective Nrf2. Astrocyte-specific Nrf2 expression induced a reactive phenotype which recapitulated elements of this proteostasis signature, reduced Aß deposition and phospho-tau accumulation in their respective models, and rescued brain-wide transcriptional deregulation, cellular pathology, neurodegeneration and behavioural/cognitive deficits. Thus, Aß and Tau induce overlapping astrocyte profiles associated with both deleterious and adaptive-protective signals, the latter of which can slow patho-progression.
Mishra D, Richard JE, Maric I, Porteiro B, Häring M, Kooijman S, Musovic S, Eerola K, López-Ferreras L, Peris E, Grycel K, Shevchouk OT, Micallef P, Olofsson CS, Wernstedt Asterholm I, Grill HJ, Nogueiras R, Skibicka KP.
PMID: 30865890 | DOI: 10.1016/j.celrep.2019.02.044
Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6.
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.
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.
Kidney international reports
Xu, K;Shang, N;Levitman, A;Corker, A;Kudose, S;Yaeh, A;Neupane, U;Stevens, J;Sampogna, R;Mills, AM;D'Agati, V;Mohan, S;Kiryluk, K;Barasch, J;
PMID: 34642645 | DOI: 10.1016/j.ekir.2021.09.005
Loss of kidney function is a common feature of COVID-19 infection, but serum creatinine (SCr) is not a sensitive or specific marker of kidney injury. We tested whether molecular biomarkers of tubular injury measured at hospital admission were associated with AKI in those with COVID-19 infection.This is a prospective cohort observational study consisting of 444 consecutive SARS-CoV-2 patients enrolled in the Columbia University Emergency Department at the peak of New York's pandemic (March-April 2020). Urine and blood were collected simultaneously at hospital admission (median time: day 0, IQR 0-2 days) and urine biomarkers analyzed by ELISA and by a novel dipstick. Kidney biopsies were probed for biomarker RNA and for histopathologic acute tubular injury (ATI) scores.Admission uNGAL was associated with AKI diagnosis (267±301 vs. 96±139 ng/mL, P < 0.0001) and staging; uNGAL levels >150ng/mL demonstrated 80% specificity and 75% sensitivity to diagnose AKI-stage 2-3. Admission uNGAL quantitatively associated with prolonged AKI, dialysis, shock, prolonged hospitalization, and in-hospital death, even when admission SCr was not elevated. The risk of dialysis increased almost 4-fold per standard deviation of uNGAL independently of baseline SCr, co-morbidities, and proteinuria [OR(95%CI): 3.59 (1.83-7.45), P < 0.001]. In COVID-19 kidneys, NGAL mRNA expression broadened in parallel with severe histopathological injury (ATI). Conversely, low uNGAL levels at admission ruled out stage 2-3 AKI (NPV 0.95, 95%CI: 0.92-0.97) and the need for dialysis (NPV: 0.98, 95%CI: 0.96-0.99)). While proteinuria and uKIM-1 implicated tubular injury, neither were diagnostic of AKI stages.In COVID-19 patients, uNGAL quantitatively associated with histopathological injury (ATI), the loss of kidney function (AKI), and the severity of patient outcomes.
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.
Bedard, MC;Chihanga, T;Carlile, A;Jackson, R;Brusadelli, MG;Lee, D;VonHandorf, A;Rochman, M;Dexheimer, PJ;Chalmers, J;Nuovo, G;Lehn, M;Williams, DEJ;Kulkarni, A;Carey, M;Jackson, A;Billingsley, C;Tang, A;Zender, C;Patil, Y;Wise-Draper, TM;Herzog, TJ;Ferris, RL;Kendler, A;Aronow, BJ;Kofron, M;Rothenberg, ME;Weirauch, MT;Van Doorslaer, K;Wikenheiser-Brokamp, KA;Lambert, PF;Adam, M;Steven Potter, S;Wells, SI;
PMID: 37031202 | DOI: 10.1038/s41467-023-37377-0
Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.
Smith RS, Kenny CJ, Ganesh V, Jang A, Borges-Monroy R, Partlow JN, Hill RS, Shin T, Chen AY, Doan RN, Anttonen AK, Ignatius J, Medne L, Bönnemann CG, Hecht JL, Salonen O, Barkovich AJ, Poduri A, Wilke M, de Wit MCY, Mancini GMS, Sztriha L, Im K, Amrom D,
PMID: 30146301 | DOI: 10.1016/j.neuron.2018.07.052
Channelopathies are disorders caused by abnormal ion channel function in differentiated excitable tissues. We discovered a unique neurodevelopmental channelopathy resulting from pathogenic variants in SCN3A, a gene encoding the voltage-gated sodium channelNaV1.3. Pathogenic NaV1.3 channels showed altered biophysical properties including increased persistent current. Remarkably, affected individuals showed disrupted folding (polymicrogyria) of the perisylvian cortex of the brain but did not typically exhibit epilepsy; they presented with prominent speech and oral motor dysfunction, implicating SCN3A in prenatal development of human cortical language areas. The development of this disorder parallels SCN3A expression, which we observed to be highest early in fetal cortical development in progenitor cells of the outer subventricular zone and cortical plate neurons and decreased postnatally, when SCN1A (NaV1.1) expression increased. Disrupted cerebral cortical folding and neuronal migration were recapitulated in ferrets expressing the mutant channel, underscoring the unexpected role of SCN3A in progenitor cells and migrating neurons.
Agnew-Svoboda, W;Ubina, T;Figueroa, Z;Wong, YC;Vizcarra, EA;Roebini, B;Wilson, EH;Fiacco, TA;Riccomagno, MM;
PMID: 36046623 | DOI: 10.1016/j.crmeth.2022.100276
Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.
Cho, I;Chang, JB;
PMID: 35233025 | DOI: 10.1038/s41598-022-06903-3
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.
