NIH - Modeling HIV Neuropathology Using Microglia from Human iPSC and Cerebral Organoids (R01, R21 Clinical Trial Not Allowed)

The following description was taken from the R01 version of this FOA.

HIV-associated neurological disorders (HAND) persist in up to 50% of HIV-patients even when HIV replication is suppressed by combination antiretroviral therapy (cART), which transformed HIV/AIDS from a fatal illness into a chronically managed long-term condition. HIV does not infect neurons, but infects microglia and macrophages in the brain, causing HAND associated neuropathology. HAND epitomizes a series of disorders include Asymptomatic Neurocognitive Impairment (ANI), Mild Neurocognitive Disorder (MND), and HIV-Associated Dementia (HAD). These neurocognitive deficits interfere with psychomotor speed and coordination, diminishing memory and executive functions, and reduce quality of life in long-standing aviremic HIV-positive patients. These clinical challenges mandate research for a better understanding of HIV neuropathology; however, currently there are no effective approaches for HIV-infected live human brain studies or realistic HIV-infected animal models for HIV neuropathology.

A growing body of evidence suggests that HIV-infected microglia and macrophages play critical roles in HIV neuropathogenesis. These infected immune cells release viral gene products, such as gp120 and tat, that are toxic to neurons and glial cells, and trigger severe immune responses through heightened secretion of cytokines and chemokines, resulting in wide-spread synaptodendritic degeneration, neuronal and glial cell losses, and neural circuit structural and functional alterations. Consistent with these findings, autopsy of HIV-infected patients and analysis of HIV/AIDS postmortem brains have shown apparent neurocircuit atrophy, with neuronal losses and gliosis, especially in the hippocampal and forebrain cortical areas. Furthermore, HIV can be in latency in the infected cells in CNS. These cells, primarily astrocytes, perivascular macrophages and microglia, can harbor replication competent virus and have become the reservoirs of the virus that hinder the eradication of HIV. This has now emerged as a major challenging area for HIV treatment, especially with the protection of blood-brain barrier and the enclosure restriction of skull cavity. The cell type specificity and responses to the virus, the variants in specific host genetic factors, the genetic variation of the evolving virus in the brain, the altering neurovirulence, neurotropism, and regional compartmentalization and trafficking in the brain, all impact the pathophysiology of HAND, and likely determine the outcome of the antiretroviral therapies.

Substance use disorders (SUD) have important roles in HIV transmission, infection, and pathology. Many of the abused substances such as opioids, cannabinoids, methamphetamine and cocaine, have been suggested to impact and alter host-virus interaction, HIV replication, immune responses, antiretroviral therapies and HAND progression. How innate and adaptive immune responses, blood-brain barrier integrity, neurotransmission and neural circuits activities, astrocyte function and CNS homeostasis are affected by abused substances, and how chronic exposure to drugs of abuse exacerbate chronical symptoms of HAND, are important research areas.

Recent technological advancements have allowed generating microglia and cerebral organoids from human induced pluripotent stem cell (iPSC) lines. The iPSC derived microglia possess the characteristics of those developed in vivo, in terms of gene transcription, migration, phagocytotic activities and responses to inflammatory stimuli, and have been shown to be fully functional in various neurological environments, secreting cytokines and phagocytosing neural debris, when introduced into cerebral organoids. Concomitantly, new advancements of 3-D culture of cerebral organoids have enabled this experimental platform for the analysis of complex neurodevelopmental processes because it recapitulates the development of complex, organ-like tissues reminiscent of those of human brain. Cerebral organoids from iPSC of neurological disease patients have been used to study cellular and molecular defects, and to image cell activities, at the single cell level in a physiologically relevant background and to test potential therapeutic compounds. The volume of the publications in high impact journals and the quality of the 3-D culture have propelled the technology to become more mature, reproducible and standardize-able.

This initiative will support projects aimed at the elucidation of microglia-neuronal pathophysiological mechanisms underlying the neuropathology of HAND, leveraging the advanced in vitro cell reprogramming technologies, 3-D culture systems and cerebral organoids derived from human iPSCs. Investigators are encouraged to exploit state of-art technologies including Omics assays, genomic editing tools such as CRISPR, and single cell analysis and imaging approaches to address outstanding questions regarding molecular regulation of persistent HIV (e.g. latency or reservoirs) in the context of chronic substance use or substance use disorders (SUD). Investigators are further encouraged to explore the lymphocyte reprograming resource at NIDA Center for Genetic Studies (contact: Dr. Jonathan Pollock, web link: https://nidagenetics.org/ ) for generating patient derived iPSC lines.

Proposed projects MUST include the following components. Applications which lack these three components will be considered non-responsive to the FOA and will not be reviewed. The major thrust of the project MUST involve exploitation of induced microglia and cerebral organoids generated from patient derived iPSC lines to better understand the molecular and cellular mechanisms of HIV-Associated Neurocognitive Disorder (HAND). At least one aim or sub-aim MUST also involve either 1. opioid, cannabinoid, methamphetamine, nicotinic, dopaminergic, or other signaling pathways relevant to addictive substance use, or 2. exposure to addictive substances, or 3. analysis of samples from patients that have used addictive substances or have SUDs. Addictive substances of interest include: opioids, nicotine, cocaine, methamphetamine, stimulants, prescription drugs, cannabinoids, alcohol, or combinations of these drugs. Applications focused solely on alcohol exposure will be considered non-responsive to this FOA.

The research areas that are pertinent to this FOA include, but are not limited to:

- Establishing iPSC lines from HIV-patients and HIV-free controls, and use these cells to generate cerebral organoids and microglial cells so that microglia can be infected by HIV and introduced into the organoids;
- Studying how HIV-infected microglia alter neural tissue structure, physiology, and function in HIV-latent, HIV-infected, and HIV-free cerebral organoids;
- Identifying the evolving mechanisms of HIV Neuropathogenesis in the chronically cART managed long-term condition, including the viral genetic or host epigenetic studies relating to the establishment and maintenance of viral latency and reservoirs in cerebral organoid tissue;
- The role of viral genetic and epigenetic factors in the pathophysiology of HAND, the role of viral and host transcriptional regulation (including viral protein modifications, histone modifications, changes in chromatin structure, and non-coding RNA) in HIV neuropathogenesis, with and without the presence of abused substances;
- The role of adaptive and innate immunity in regulating anti-viral responses in cerebral organoid tissues, and the effects of abused substances on this regulation;
- Host gene regulation in neuronal and glial cells following exposure to exosomes released from HIV-infected microglial cells and astrocytes, with and without the presence of abused substances;
- The novel cellular, molecular and cell imaging markers of chronic HIV-associated neurological changes, with and without the exposure to the abused substances;
- The genomics, proteomics, metabolomics, systems biology and neuroimaging of microglia and host organoid tissues, compared with what is discovered from in vivo models and animal studies;
- Processes and pathways of inflammatory mediators, viral factors, excitotoxicity and signaling mechanisms leading to neuronal toxicity by active and latent HIV with cART maintenance in cerebral organoids;
- Synaptodendritic degenerative changes and neuronal and glial cell losses in the setting of chronic HIV-infection, and the protective vs. pathogenic effects of those changes with and without the HIV-infected microglia, analyzed at the single cell level;
- Changes in neuronal excitability, synaptic plasticity, and neural circuit activity that contribute to the development of neuropathology of HAND, with and without the HIV-infected microglia, and with and without the presence of abused substances, analyzed at the single cell level;
- The impact of substances of abuse on the cellular and molecular mechanisms associated with the neuropathology underlying HAND, including HIV trafficking in neural tissues, neuron-glial communication, cell signaling, membrane traffic, protein chemistry and neurophysiologic changes and excitability, and neuroplasticity;
- Genetic and genomic editing, aided by the state-of-art technologies such as CRISPR, to alleviate the cellular and molecular disorders associated with the neuropathology underlying HAND;
- Genetic and genomic editing, aided by the state-of-art technologies such as CRISPR, to eradicate the virus in HIV-infected neural tissues such as cerebral organoids, especially the viral factors in HIV-infected microglia, in the setting of chronic HIV-infection and cART maintenance, with and without the presence of abused substances.

Deadline: November 18, 2018 (letters of intent); December 18, 2018 (full proposals)

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NIH – Modeling HIV Neuropathology Using Microglia from Human iPSC and Cerebral Organoids (R01, R21 Clinical Trial Not Allowed)