Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as ...such by their expression of glial fibrillary acidic protein, glutamine synthetase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathognomonic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9+ cells with GLT1+ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple mini-strokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immunolabeling. The analysis showed that SOX9+ astrocytes constitute ∼10-20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain.
Astrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain.
An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of ...this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (hipk) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole Caenorhabditis elegans Hipk homolog, hpk-1, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of hpk-1 is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that hpk-1 is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors pha-4/FoxA and mxl-2/Mlx, but not hlh-30/TFEB or the nuclear hormone receptor nhr-62, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.
Lifespan is the most straightforward surrogate measure of aging, as it is easily quantifiable. A common approach to measure Caenorhabditis elegans lifespan is to follow a population of animals over ...time and score viability based on movement. We previously developed an alternative approach, called the Replica Set method, to quantitatively measure lifespan of C. elegans in a high-throughput manner. The replica set method allows a single investigator to screen more treatments or conditions in the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. Unlike traditional approaches, the Replica Set method centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with "traditional" longitudinal methods. The protocols provided here describe both the traditional experimental approach and the Replica Set method, as well as practical considerations for each.
Huntington’s disease (HD) is characterized by hypomyelination and neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic ...stem cells (hESCs) derived from mutant Huntingtin (mHTT) embryos or normal controls and performed RNA sequencing (RNA-seq) to assess mHTT-dependent changes in gene expression. In human GPCs (hGPCs) derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply downregulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF, and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell-autonomous, mHTT-dependent suppression of glial differentiation.
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•Mutant HTT-expressing hESCs exhibit a relative block in glial lineage progression•Differentiation-associated genes are downregulated in mHTT glial progenitor cells•Human glial chimeras established with mHTT glial progenitors are hypomyelinated•mHTT human glial chimeras manifest disrupted astrocytic differentiation
The authors show that glial progenitor cells derived from human ESCs expressing mutant Huntingtin (mHTT) are delayed and defective in their maturation; their suppressed transcription of differentiation-associated genes leads to both astrocytic dysfunction and myelin deficiency in vivo. Glial pathology may thus contribute to disease phenotype in Huntington’s disease.
Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under ...fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered the
homeodomain-interacting protein kinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss of
results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aging
expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system,
induction overlaps with key longevity transcription factors, which suggests
expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression of
extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requires
(MLX),
(TFEB), and
(FOXO). Our work establishes
as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.
Glial pathology is a causal contributor to the striatal neuronal dysfunction of Huntington’s disease (HD). We investigate mutant HTT-associated changes in gene expression by mouse and human striatal ...astrocytes, as well as in mouse microglia, to identify commonalities in glial pathobiology across species and models. Mouse striatal astrocytes are fluorescence-activated cell sorted (FACS) from R6/2 and zQ175 mice, which respectively express exon1-only or full-length mHTT, and human astrocytes are generated either from human embryonic stem cells (hESCs) expressing full-length mHTT or from fetal striatal astrocytes transduced with exon1-only mHTT. Comparison of differential gene expression across these conditions, all with respect to normal HTT controls, reveals cell-type-specific changes in transcription common to both species, yet with differences that distinguish glia expressing truncated mHTT versus full-length mHTT. These data indicate that the differential gene expression of glia expressing truncated mHTT may differ from that of cells expressing full-length mHTT, while identifying a conserved set of dysregulated pathways in HD glia.
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•Glial transcription differs between cells expressing full-length and exon1-only mHTT•Truncated mHTT inhibits glial cholesterol pathway expression; full-length mHTT does not•Astrocytic structural genes are dysregulated by mHTT-expressing glia in all models•Mutant HTT-expressing mouse astrocytes manifest altered fiber distributions in vivo
Benraiss et al. assess astrocytic and microglial gene expression across mouse and human models of Huntington’s disease, to define commonalities that may contribute to HD pathogenesis. They report differences between glia expressing full-length and exon 1-only mHTT and identify a core set of dysregulated pathways that predict glial pathology.
The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For ...instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington's disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.
Dietary restriction (DR), the process of decreasing overall food consumption over an extended period of time, has been shown to increase longevity across evolutionarily diverse species and delay the ...onset of age-associated diseases in humans. In Caenorhabditis elegans, the Myc-family transcription factors (TFs) MXL-2 (Mlx) and MML-1 (MondoA/ChREBP), which function as obligate heterodimers, and PHA-4 (orthologous to FOXA) are both necessary for the full physiological benefits of DR. However, the adaptive transcriptional response to DR and the role of MML-1::MXL-2 and PHA-4 remains elusive. We identified the transcriptional signature of C. elegans DR, using the eat-2 genetic model, and demonstrate broad changes in metabolic gene expression in eat-2 DR animals, which requires both mxl-2 and pha-4. While the requirement for these factors in DR gene expression overlaps, we found many of the DR genes exhibit an opposing change in relative gene expression in eat-2;mxl-2 animals compared to wild-type, which was not observed in eat-2 animals with pha-4 loss. Surprisingly, we discovered more than 2000 genes synthetically dysregulated in eat-2;mxl-2, out of which the promoters of down-regulated genes were substantially enriched for PQM-1 and ELT-1/3 GATA TF binding motifs. We further show functional deficiencies of the mxl-2 loss in DR outside of lifespan, as eat-2;mxl-2 animals exhibit substantially smaller brood sizes and lay a proportion of dead eggs, indicating that MML-1::MXL-2 has a role in maintaining the balance between resource allocation to the soma and to reproduction under conditions of chronic food scarcity. While eat-2 animals do not show a significantly different metabolic rate compared to wild-type, we also find that loss of mxl-2 in DR does not affect the rate of oxygen consumption in young animals. The gene expression signature of eat-2 mutant animals is consistent with optimization of energy utilization and resource allocation, rather than induction of canonical gene expression changes associated with acute metabolic stress, such as induction of autophagy after TORC1 inhibition. Consistently, eat-2 animals are not substantially resistant to stress, providing further support to the idea that chronic DR may benefit healthspan and lifespan through efficient use of limited resources rather than broad upregulation of stress responses, and also indicates that MML-1::MXL-2 and PHA-4 may have distinct roles in promotion of benefits in response to different pro-longevity stimuli.Dietary restriction (DR), the process of decreasing overall food consumption over an extended period of time, has been shown to increase longevity across evolutionarily diverse species and delay the onset of age-associated diseases in humans. In Caenorhabditis elegans, the Myc-family transcription factors (TFs) MXL-2 (Mlx) and MML-1 (MondoA/ChREBP), which function as obligate heterodimers, and PHA-4 (orthologous to FOXA) are both necessary for the full physiological benefits of DR. However, the adaptive transcriptional response to DR and the role of MML-1::MXL-2 and PHA-4 remains elusive. We identified the transcriptional signature of C. elegans DR, using the eat-2 genetic model, and demonstrate broad changes in metabolic gene expression in eat-2 DR animals, which requires both mxl-2 and pha-4. While the requirement for these factors in DR gene expression overlaps, we found many of the DR genes exhibit an opposing change in relative gene expression in eat-2;mxl-2 animals compared to wild-type, which was not observed in eat-2 animals with pha-4 loss. Surprisingly, we discovered more than 2000 genes synthetically dysregulated in eat-2;mxl-2, out of which the promoters of down-regulated genes were substantially enriched for PQM-1 and ELT-1/3 GATA TF binding motifs. We further show functional deficiencies of the mxl-2 loss in DR outside of lifespan, as eat-2;mxl-2 animals exhibit substantially smaller brood sizes and lay a proportion of dead eggs, indicating that MML-1::MXL-2 has a role in maintaining the balance between resource allocation to the soma and to reproduction under conditions of chronic food scarcity. While eat-2 animals do not show a significantly different metabolic rate compared to wild-type, we also find that loss of mxl-2 in DR does not affect the rate of oxygen consumption in young animals. The gene expression signature of eat-2 mutant animals is consistent with optimization of energy utilization and resource allocation, rather than induction of canonical gene expression changes associated with acute metabolic stress, such as induction of autophagy after TORC1 inhibition. Consistently, eat-2 animals are not substantially resistant to stress, providing further support to the idea that chronic DR may benefit healthspan and lifespan through efficient use of limited resources rather than broad upregulation of stress responses, and also indicates that MML-1::MXL-2 and PHA-4 may have distinct roles in promotion of benefits in response to different pro-longevity stimuli.
People living with HIV are at higher risk of atherosclerosis (AS). The pathogenesis of this risk is not fully understood. To assess the regulatory networks involved in AS we sequenced mRNA of the ...peripheral blood mononuclear cells (PBMCs) and measured cytokine and chemokine levels in the plasma of 13 persons living with HIV and 12 matched HIV-negative persons with and without AS. microRNAs (miRNAs) are known to play a role in HIV infection and may modulate gene regulation to drive AS. Hence, we further assessed miRNA expression in PBMCs of a subset of 12 HIV+ people with and without atherosclerosis. We identified 12 miRNAs differentially expressed between HIV+ AS+ and HIV+ , and validated 5 of those by RT-qPCR. While a few of these miRNAs have been implicated in HIV and atherosclerosis, others are novel. Integrating miRNA measurements with mRNA, we identified 27 target genes including SLC4A7, a critical sodium and bicarbonate transporter, that are potentially dysregulated during atherosclerosis. Additionally, we uncovered that levels of plasma cytokines were associated with transcription factor activity and miRNA expression in PBMCs. For example, BACH2 activity was associated with IL-1β, IL-15, and MIP-1α. IP10 and TNFα levels were associated with miR-124-3p. Finally, integration of all data types into a single network revealed increased importance of miRNAs in network regulation of the HIV+ group in contrast with increased importance of cytokines in the HIV+ AS+ group.
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