Faculty Bibliography

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1alpha, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.

The decline of cognitive function occurs with aging, but the mechanisms responsible are unknown. Astrocytes instruct the formation, maturation, and elimination of synapses, and impairment of these functions has been implicated in many diseases. These findings raise the question of whether astrocyte dysfunction could contribute to cognitive decline in aging. We used the Bac-Trap method to perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse. We found that astrocytes have region-specific transcriptional identities that change with age in a region-dependent manner. We validated our findings using fluorescence in situ hybridization and quantitative PCR. Detailed analysis of the differentially expressed genes in aging revealed that aged astrocytes take on a reactive phenotype of neuroinflammatory A1-like reactive astrocytes. Hippocampal and striatal astrocytes up-regulated a greater number of reactive astrocyte genes compared with cortical astrocytes. Moreover, aged brains formed many more A1 reactive astrocytes in response to the neuroinflammation inducer lipopolysaccharide. We found that the aging-induced up-regulation of reactive astrocyte genes was significantly reduced in mice lacking the microglial-secreted cytokines (IL-1α, TNF, and C1q) known to induce A1 reactive astrocyte formation, indicating that microglia promote astrocyte activation in aging. Since A1 reactive astrocytes lose the ability to carry out their normal functions, produce complement components, and release a toxic factor which kills neurons and oligodendrocytes, the aging-induced up-regulation of reactive genes by astrocytes could contribute to the cognitive decline in vulnerable brain regions in normal aging and contribute to the greater vulnerability of the aged brain to injury.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-beta pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-alpha (TNF-alpha) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-alpha. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an epsilon4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-beta pathology with symptomatic Alzheimer disease who usually have tau pathology, epsilon4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-beta pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

Perinatal white matter injury (WMI) is the leading cause of long-term neurological morbidity in infants born preterm. Neuroinflammation during a critical window of early brain development plays a key role in WMI disease pathogenesis. The mechanisms linking inflammation with the long-term myelination failure that characterizes WMI, however, remain unknown. Here, we investigate the role of astrocyte reactivity in WMI. In an experimental mouse model of WMI, we demonstrate that WMI disease outcomes are improved in mutant mice lacking secretion of inflammatory molecules TNF-α, IL-1α, and C1q known, in addition to other roles, to induce the formation of a neuroinflammatory reactive astrocyte substate. We show that astrocytes express molecular signatures of the neuroinflammatory reactive astrocyte substate in both our WMI mouse model and human tissue affected by WMI, and that this gene expression pattern is dampened in injured mutant mice. Our data provide evidence that a neuroinflammatory reactive astrocyte substate correlates with adverse WMI disease outcomes, thus highlighting the need for further investigation of these cells as potential causal players in WMI pathology.

In addition to their many functions in the healthy central nervous system (CNS), astrocytes respond to CNS damage and disease through a process called "reactivity." Recent evidence reveals that astrocyte reactivity is a heterogeneous spectrum of potential changes that occur in a context-specific manner. These changes are determined by diverse signaling events and vary not only with the nature and severity of different CNS insults but also with location in the CNS, genetic predispositions, age, and potentially also with "molecular memory" of previous reactivity events. Astrocyte reactivity can be associated with both essential beneficial functions as well as with harmful effects. The available information is rapidly expanding and much has been learned about molecular diversity of astrocyte reactivity. Emerging functional associations point toward central roles for astrocyte reactivity in determining the outcome in CNS disorders.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNβ and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence during the development of the brain and spinal cord. Here we used single-cell/single-nucleus RNA sequencing (scRNA-seq/snRNA-seq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human-induced pluripotent stem cells. We computationally identify candidate genes involved in the fate specification of glia in both species and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our transcriptomic data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multi-omic, dual single-nuclei (sn)RNA-seq/snATAC-seq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets will enable future optimization of glial differentiation protocols and provide insight into human glial differentiation.

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.