Kelly L. Jordan-Sciutto

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK-B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK-A, the major PERK haplotype, and PERK-B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK-A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK-B exhibited similar levels of eIF2α phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4–6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low-dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Differentiating oligodendrocytes undergo dramatic morphologic alterations to transition from progenitors to mature oligodendrocytes that synthesize myelin, the lipid-rich membrane coating axons which strengthens saltatory conduction and provides metabolic support. Actin dynamics, which are often regulated by membrane bound nucleators associated with organelles, underpin the morphologic shifts in oligodendrocyte maturation; however, the origin of such regulation during oligodendrocyte differentiation remains unknown. Here, we demonstrate that the lysosomal non-selective cation channel, transient potential mucolipin 1 (TRPML1), is a critical regulator of oligodendrocyte morphology during differentiation and initial myelination. Lysosomes move into oligodendrocyte processes during differentiation. While manipulation of TRPML1 did not change the expression of oligodendrocyte lineage markers, activation of TRPML1 resulted in altered oligodendrocyte morphology and an increase in actin filament content driven by the small GTPase Rac1 and subsequent disinhibition of PAK1 via phosphorylation. Actin associated changes in morphology are accompanied by the presence of lysosomal-derived calcium transients in nascent oligodendrocyte processes, potentially revealing a link between localized calcium signaling and actin polymerization. Lastly, adolescent mice ( Mcoln1 ^-/- ), in which TRPML1 had been deleted, had significantly impaired myelination and decreased numbers of mature oligodendrocyte, which was associated with a reduction in staining for the phosphorylated form of the actin regulator, PAK1, in the motor cortex and corpus callosum as evidence of decreased TRPML1/Rac1/PAK1 signaling. Together, our work reveals lysosomal TRPML1 activity as a central regulator of oligodendrocyte morphology independent of myelin protein expression and may provide mechanistic insight into the distinct but coordinated pathways that lead to oligodendrocyte differentiation and how lysosomal dysfunction impacts these processes in diseased states.

Despite significant advances in medical treatment, human immunodeficiency virus 1 (HIV-1) infection continues to be a global healthcare burden. Approximately 40 million people worldwide are living ...

People living with HIV suffer multiple comorbid conditions related to chronic inflammation at increased rates compared to the general population, even when on effective antiretroviral therapy. In particular, current data indicate that the increased incidence and severity of neurocognitive impairment (NCI) are associated with unresolved neuroinflammation. Attempts to treat NCI in people living with HIV by reducing inflammation have thus far been unsuccessful, suggesting that a more mechanistic understanding of inflammatory processes in the CNS during HIV is necessary. Here, we use iPSC-derived microglia (iMg) and astrocytes (iAst) to model HIV infection in the CNS. We show that our iMg robustly express markers associated with microglial identity and are susceptible to HIV infection, but exhibit lower HIV replication rates and weaker immune response to HIV challenge compared to monocyte-derived macrophages. Coculture of iAst with iMg leads to a much stronger pro-inflammatory immune response, and, surprisingly, a robust increase in rates of HIV replication. Increased replication in iMg/iAst cocultures is associated with higher levels of multiple pro-inflammatory cytokines, including TNFα, which is produced by iAst upon exposure to HIV-infected iMg. Addition of exogenous TNFα to iMg during HIV infection is also sufficient to increase rates of replication, and neutralization of TNFα via adalimumab/Humira treatment in iMg/iAst cocultures reduces replication. Blocking NF-kB signaling with iKK inhibitor Bay-11-7082 (Bay-11) demonstrates that increased HIV replication in iMg/iAst cocultures is due to increased NF-kB activity. Finally, we show that in HIV-infected iMg there is movement of lysosomes to the periphery of the cell membrane and release of lysosomal content into the extracellular space, suggesting that this dysregulated lysosomal flux could further contribute to the pro-inflammatory microenvironment. We propose that this altered lysosomal trafficking and increased cytokine production drives a pro-inflammatory phenotype in glia and represents a potential source of unresolved neuroinflammation in people living with HIV.

Emerging evidence suggests brain-resident myeloid cells, including perivascular macrophages and microglia, provide a reservoir for HIV infection in the central nervous system (CNS), and their inflammatory activation is a proposed pathogenic mechanism in HIV-associated neurocognitive disorders (HAND). We investigated whether cannabinoid receptor 2 (CB₂), an immunomodulatory receptor expressed in myeloid cells, regulates viral replication and inflammation in HIV-infected macrophages and microglia. Using the synthetic CB₂-specific agonist JWH-133, we found that CB₂ activation reduced HIV replication in primary human monocyte-derived macrophages (MDMs) and human induced pluripotent stem cell-derived microglia (iMg) at differing doses, corresponding to the basal expression of CNR2, which encodes CB₂, and related endocannabinoid transcripts in each cell type. JWH-133 broadly reduced release of cytokines from HIV-infected MDMs but not iMg. RNA-seq revealed that CB₂ agonism primarily altered interferon and integrated stress response pathways in MDMs while altering homeostatic pathways, including synapse maintenance and phagocytosis, in iMg. Further analyses in iMg revealed that NLRP3 inflammasome activation, but not priming, was reduced by CB₂ activation, which did not inhibit HIV-induced nuclear factor kB activation. This study identifies key differences in CB₂ response between myeloid lineage cell types and implicates CB₂-specific agonists as promising candidates for the regulation of HIV-associated neuroinflammation. Graphical Abstract Created in BioRender. Espinoza, C. (2025) https://BioRender.com/mxfla3i

People living with HIV suffer multiple comorbid conditions related to chronic inflammation at increased rates compared to the general population, even when on effective antiretroviral therapy. In particular, current data indicate that the increased incidence and severity of neurocognitive impairment (NCI) are associated with unresolved neuroinflammation. Attempts to treat NCI in people living with HIV by reducing inflammation have thus far been unsuccessful, suggesting that a more mechanistic understanding of inflammatory processes in the CNS during HIV is necessary. Here, we use iPSC-derived microglia (iMg) and astrocytes (iAst) to model HIV infection in the CNS. We show that our iMg robustly express markers associated with microglial identity and are susceptible to HIV infection, but exhibit lower HIV replication rates and weaker immune response to HIV challenge compared to monocyte-derived macrophages. Coculture of iAst with iMg leads to a much stronger pro-inflammatory immune response, and, surprisingly, a robust increase in rates of HIV replication. Increased replication in iMg/iAst cocultures is associated with higher levels of multiple pro-inflammatory cytokines, including TNFα, which is produced by iAst upon exposure to HIV-infected iMg. Addition of exogenous TNFα to iMg during HIV infection is also sufficient to increase rates of replication, and neutralization of TNFα via adalimumab/Humira treatment in iMg/iAst cocultures reduces replication. Blocking NF-kB signaling with iKK inhibitor Bay-11-7082 (Bay-11) demonstrates that increased HIV replication in iMg/iAst cocultures is due to increased NF-kB activity. Finally, we show that in HIV-infected iMg there is movement of lysosomes to the periphery of the cell membrane and release of lysosomal content into the extracellular space, suggesting that this dysregulated lysosomal flux could further contribute to the pro-inflammatory microenvironment. We propose that this altered lysosomal trafficking and increased cytokine production drives a pro-inflammatory phenotype in glia and represents a potential source of unresolved neuroinflammation in people living with HIV.

A disproportionate percentage of adolescents are diagnosed with human immunodeficiency virus (HIV) in the United States each year. Preexposure prophylaxis (PrEP), an antiretroviral regimen, is effective at preventing the transmission of HIV to adolescents at substantial risk for acquiring HIV. However, other select antiretrovirals have been shown to cause white matter deficits in experimental models. Adolescents taking PrEP are uniquely vulnerable to myelin impairments as the adolescent brain undergoes high rates of myelination. Here, we report that PrEP significantly reduced oligodendrocyte maturation in adolescent rats. Furthermore, cultures of primary rat oligodendrocyte progenitors treated with PrEP showed inhibited oligodendrocyte differentiation through deacidification of lysosomes resulting in lysosomal accumulation of myelin proteins. Acidic nanoparticle co‐administration with PrEP prevented PrEP‐induced oligodendrocyte maturation impairments both in vivo and in vitro. These studies suggest uninfected adolescents are vulnerable to PrEP‐induced oligodendrocyte impairments and identify maintenance of lysosome pH as a critical factor in antiretroviral design.