Faculty Bibliography

Alzheimer's disease (AD) is the most common form of dementia. Despite many years of intense research, there is currently still no effective treatment. Multiple cell types contribute to disease pathogenesis, with an increasing body of data pointing to the active participation of astrocytes. Astrocytes play a pivotal role in the physiology and metabolic functions of neurons and other cells in the central nervous system. Because of their interactions with other cell types, astrocyte functions must be understood in their biologic context, thus many studies have used mouse models, of which there are over 190 available for AD research. However, none appear able to fully recapitulate the many functional changes in astrocytes reported in human AD brains. Our review summarizes the observations of astrocyte biology noted in mouse models of familial and sporadic AD. The limitations of AD mouse models will be discussed and current attempts to overcome these disadvantages will be described. With increasing understanding of the non-neuronal contributions to disease, the development of new methods and models will provide further insights and address important questions regarding the roles of astrocytes and other non-neuronal cells in AD pathophysiology. The next decade will prove to be full of exciting opportunities to address this devastating disease.

Microglia-astrocyte interactions represent a delicate balance affecting neural cell functions in health and disease. Tightly controlled to maintain homeostasis during physiological conditions, rapid and prolonged departures during disease, infection, and following trauma drive multiple outcomes: both beneficial and detrimental. Recent sequencing studies at the bulk and single-cell level in humans and rodents provide new insight into microglia-astrocyte communication in homeostasis and disease. However, the complex changing ways these two cell types functionally interact has been a barrier to understanding disease initiation, progression, and disease mechanisms. Single cell sequencing is providing new insights; however, many questions remain. Here, we discuss how to bridge transcriptional states to specific functions so we can develop therapies to mediate negative effects of altered microglia-astrocyte interactions.

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f⁺ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f⁺ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f⁺ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.

Here we demonstrate a technique to generate proteomic signatures of specific cell types within heterogeneous populations. While our method is broadly applicable across biological systems, we have limited the current work to study neural cell types isolated from human, post-mortem Alzheimer's disease (AD) and aged-matched non-symptomatic (NS) brains. Motivating the need for this tool, we conducted an initial meta-analysis of current, human AD proteomics studies. While the results broadly corroborated major neurodegenerative disease hypotheses, cell type-specific predictions were limited. By adapting our Formaldehyde-fixed Intracellular Target-Sorted Antigen Retrieval (FITSAR) method for proteomics and applying this technique to characterize AD and NS brains, we generated enriched neuron and astrocyte proteomic profiles for a sample set of donors (available at www.fitsarpro.appspot.com). Results showed the feasibility for using FITSAR to evaluate cell-type specific hypotheses. Our overall methodological approach provides an accessible platform to determine protein presence in specific cell types and emphasizes the need for protein-compatible techniques to resolve systems complicated by cellular heterogeneity.

Reactive astrocytes have been implicated in the pathogenesis of neurodegenerative diseases, including a non-cell autonomous effect on motor neuron survival in ALS. We previously defined a mechanism by which microglia release three factors, IL-1α, TNFα, and C1q, to induce neurotoxic astrocytes. Here we report that knocking out these three factors markedly extends survival in the SOD1^G93A ALS mouse model, providing evidence for gliosis as a potential ALS therapeutic target.

The COVID19 crisis has magnified the issues plaguing academic science, but it has also provided the scientific establishment with an unprecedented opportunity to reset. Shoring up the foundation of academic science will require a concerted effort between funding agencies, universities, and the public to rethink how we support scientists, with a special emphasis on early career researchers.

Glaucoma is a neurodegenerative disease that features the death of retinal ganglion cells (RGCs) in the retina, often as a result of prolonged increases in intraocular pressure. We show that preventing the formation of neuroinflammatory reactive astrocytes prevents the death of RGCs normally seen in a mouse model of glaucoma. Furthermore, we show that these spared RGCs are electrophysiologically functional and thus still have potential value for the function and regeneration of the retina. Finally, we demonstrate that the death of RGCs depends on a combination of both an injury to the neurons and the presence of reactive astrocytes, suggesting a model that may explain why reactive astrocytes are toxic only in some circumstances. Altogether, these findings highlight reactive astrocytes as drivers of RGC death in a chronic neurodegenerative disease of the eye.

Regulating gene expression at the protein level is becoming increasingly important for answering basic questions in neurobiology. Several techniques using destabilizing domains (DD) on transgenes, which can be activated or deactivated by specific drugs, have been developed to achieve this goal. A DD from bacterial dihydrofolate reductase bound and stabilized by trimethoprim (TMP) represents such a tool. To control transgenic protein levels in the brain, the DD-regulating drugs need to have sufficient penetration into the central nervous system (CNS). Yet, very limited information is available on TMP pharmacokinetics in the CNS following systemic injection. Here, we performed a pharmacokinetic study on the penetration of TMP into different CNS compartments in the rat. We used mass spectrometry to measure TMP concentrations in serum, cerebrospinal fluid (CSF) and tissue samples of different CNS regions upon intraperitoneal TMP injection. We show that TMP quickly (within 10 min) penetrates from serum to CSF through the blood-CSF barrier. TMP also shows quick penetration into brain tissue but concentrations were an order of magnitude lower compared to serum or CSF. TMP concentration in spinal cord was lower than in any other analyzed CNS area. Nevertheless, effective levels of TMP to stabilize DDs can be reached in the CNS with half-lives around 2 h. These data show that TMP has good and fast penetration properties into the CNS and is therefore a valuable ligand for precisely controlling protein expression in the CNS in rodents.

In neurodegenerative diseases, debris of dead neurons are thought to trigger glia-mediated neuroinflammation, thus increasing neuronal death. Here we show that the expression of neurotoxic proteins associated with these diseases in microglia alone is sufficient to directly trigger death of naive neurons and to propagate neuronal death through activation of naive astrocytes to the A1 state. Injury propagation is mediated, in great part, by the release of fragmented and dysfunctional microglial mitochondria into the neuronal milieu. The amount of damaged mitochondria released from microglia relative to functional mitochondria and the consequent neuronal injury are determined by Fis1-mediated mitochondrial fragmentation within the glial cells. The propagation of the inflammatory response and neuronal cell death by extracellular dysfunctional mitochondria suggests a potential new intervention for neurodegeneration-one that inhibits mitochondrial fragmentation in microglia, thus inhibiting the release of dysfunctional mitochondria into the extracellular milieu of the brain, without affecting the release of healthy neuroprotective mitochondria.