Brendan Rooney

Institute for Neurodegenerative Disorders

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Kun Leng

University of California, San Francisco

Martin Kampmann

University of California, San Francisco

Weisheng Wang

George Washington University

Wenlong Xia

University of California, San Francisco

Priscilla A. Furth

Georgetown University

Vinona Muralidaran

Georgetown University Medical Center

Stephen P.J. Fancy

University of California, San Francisco

Indigo V.L. Rose

University of California, San Francisco

Marcus Y. Chin

Denali Therapeutics (United States)

Aimee W. Kao

University of California, San Francisco

Cooperative Institutions

University of California, San Francisco

Harvard University

Broad Institute

University of Eastern Finland

Georgetown University

Finland University

Kuopio University Hospital

Chan Zuckerberg Initiative (United States)

Stanley Center for Psychiatric Research

University Memory and Aging Center

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mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Kun Leng Brendan Rooney Frank McCarthy Wenlong Xia Indigo V.L. Rose

ABSTRACT Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo – IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states, was upregulated in astrocytes in multiple sclerosis lesions, and preferentially co-localized with astrocytes in hypoxic-ischemic encephalopathy. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

10.1101/2021.09.11.459904

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Citations (9)

Early Alzheimer’s disease pathology in human cortex involves transient cell states

Cell (2023)

Vahid Gazestani Tushar Kamath Naeem Nadaf Antonios Dougalis S.J. Burris

10.1016/j.cell.2023.08.005

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mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes

Journal of Neuroinflammation (2024)

Kun Leng Brendan Rooney Frank McCarthy Wenlong Xia Indigo V.L. Rose

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

Exosome

10.1186/s12974-024-03165-w

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Citations (4)

Early Alzheimer’s disease pathology in human cortex is associated with a transient phase of distinct cell states

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Vahid Gazestani Tushar Kamath Naeem Nadaf S.J. Burris Brendan Rooney

Cellular perturbations underlying Alzheimer's disease are primarily studied in human postmortem samples and model organisms. Here we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of Alzheimer's disease pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the Early Cortical Amyloid Response-were prominent in neurons, wherein we identified a transient state of hyperactivity preceding loss of excitatory neurons, which correlated with the selective loss of layer 1 inhibitory neurons. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathological burden increased. Lastly, both oligodendrocytes and pyramidal neurons upregulated genes associated with amyloid beta production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Pathogenesis

10.1101/2023.06.03.543569

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Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model

ACS Chemical Neuroscience (2022)

Marcus Y. Chin Kean-Hooi Ang Julia L. Davies Carolina Alquézar Virginia Garda

Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

High-Content Screening

Phenotypic screening

High-Throughput Screening

10.1021/acschemneuro.1c00804

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CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Kun Leng Indigo V.L. Rose Hyosung Kim Wenlong Xia Wilber Romero-Fernández

ABSTRACT In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states and functional outputs. Certain inflammatory insults induce reactive astrocytes that lose homeostatic functions and gain harmful outputs through cellular pathways that are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures – one promoted by and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in the human brain in Alzheimer’s disease and hypoxic ischemic encephalopathy. Furthermore, we validated that these signatures were regulated by Stat3 in vivo. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

10.1101/2021.08.23.457400

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Citations (17)

Table of Contents

American Journal Of Pathology (2022)

Plascencia- Germán George Perry Yingtong Dou Theresa T. Pizarro Lan Zhou

Table (database)

10.1016/s0002-9440(22)00262-0

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Esr1 but Not CYP19A1 Overexpression in Mammary Epithelial Cells during Reproductive Senescence Induces Pregnancy-Like Proliferative Mammary Disease Responsive to Anti-Hormonals

American Journal Of Pathology (2022)