Cagla Akay

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.

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.

Summary: PERK is a kinase that, in response to ER stress, mediates duplicitous homeostatic and pro-apoptotic signaling. Thus, intricate regulation is required for physiologic function. Attempts to modulate PERK activity have shown that the determinants of adaptive vs. mal-adaptive signaling remain ambiguous. Here, with purified protein, we provide direct evidence that PERK binds copper, identify the residues that coordinate binding, and demonstrate that binding is necessary for kinase activity. Furthermore cellular PERK activity can be modulated via copper availability, and this regulatory relationship can be manipulated to dictate ER stress tolerance. Critically, these phenomenon translate to phenotypes in vivo , as C. elegans harboring a “copper-binding mutant” of PERK exhibit enhanced ER-stress sensitivity. The copper-PERK paradigm adds onto a new class of dynamic copper-binding enzymes and implicates that copper homeostasis, as a regulator of PERK, may constitute a previously unknown variable to resolve long-standing ambiguity in endeavors to therapeutically target PERK.

Coding and noncoding single-nucleotide variants (SNVs) of EIF2AK3 , which encodes an integrated stress response (ISR) kinase, may play a role in neurodegenerative disorders. We used a candidate gene approach to determine the correlation of EIF2AK3 SNVs with neurocognitive (NC) impairment (NCI), which can persist with viral suppression from antiretroviral therapy (ART) in people with HIV (PWH). This retrospective study of prospectively collected data included participants of the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort, after excluding participants with severe neuropsychiatric comorbidities. Genome-wide data previously obtained in the CHARTER cohort participants (n=1,047) were analyzed to interrogate the association of three noncoding EIF2AK3 SNVs with the continuous global deficit score (GDS) and global NCI (GDS≥0.5). Targeted sequencing (TS) was performed in 992 participants with available genomic DNA to determine the association of three coding EIF2AK3 SNVs with GDS and NCI. Analyses included univariable and multivariable methods such as analysis of variance and regression. Multivariable models covaried demographic, disease-associated, and treatment characteristics. The cohort characteristics were as follows: median age, 43.1 years; females, 22.8%; European ancestry, 41%; median CD4+ T cell counts, 175/µL (nadir) and 428/µL (current). At first assessment, 70.5% used ART and 68.3% of these had plasma HIV RNA ≤ 200 copies/mL. A minority of participants had at least one risk allele for rs6739095 (T,41.7%), rs1913671 (C,41.4%), and rs11684404 (C,39.4%). All three noncoding EIF2AK3 SNVs were associated with significantly worse GDS and more NCI (all p p 13 were independently associated with GDS and NCI ( p EIF2AK3 SNVs were specifically associated with worse performance in executive functioning, motor functioning, learning, and verbal fluency. Coding and non-coding SNVs of EIF2AK3 were associated with global NC and domain-specific performance. The effects were small-to-medium in size but were present in multivariable analyses. Specific SNVs in EIF2AK3 may be an important component of genetic vulnerability to NC complications in PWH. Identification of host factors that predict NCI could allow for earlier interventions, including those directly modulating the ISR, to improve NC outcomes.

Despite combined antiretroviral therapy (cART), HIV‐associated neurocognitive disorder (HAND) affects 30–50% of HIV‐positive patients. Importantly, persistent white matter pathologies, specifically corpus callosum thinning and disruption of white matter microstructures observed in patients with HAND despite viral control through cART, raise the possibility that HIV infection in the setting of suboptimal cART may perturb oligodendrocyte (OL) maturation, function and/or survival, influencing HAND persistence in the cART era. To examine the effect of HIV infection on OL maturation, we used supernatants of primary human monocyte‐derived macrophages infected with HIV (HIV/MDMs) to treat primary cultures of rat oligodendrocyte precursor cells (OPCs) during their differentiation to mature OLs. Using immunostaining for lineage‐specific markers, we found that HIV/MDMs significantly inhibited OPC maturation. Based on our previous studies, we examined the potential role of several signaling pathways, including ionotropic glutamate receptors and the integrated stress response (ISR), and found that AMPA receptors (AMPAR)/kainic acid (KA) receptors (KARs) mediated the HIV/MDMs‐induced defect in OL maturation. We also found that the treatment of OPC cultures with glutamate or AMPAR/KAR agonists phenocopied this effect. Blocking ISR activation, specifically the PERK arm of the ISR, protected OPCs from HIV/MDMs‐mediated inhibition of OL maturation. Further, while glutamate, AMPA, and KA activated the ISR, inhibition of AMPAR/KAR activation prevented ISR induction in OPCs and rescued OL maturation. Collectively, these data identify glutamate signaling via ISR activation as a potential therapeutic pathway to ameliorate white matter pathologies in HAND and highlight the need for further investigation of their contribution to cognitive impairment.

The proposed molecular mechanisms underlying neurodegenerative pathogenesis are varied, precluding the development of effective therapies for these increasingly prevalent disorders. One of the most consistent observations across neurodegenerative diseases is the phosphorylation of eukaryotic initiation factor 2α (eIF2α). eIF2α is a translation initiation factor, involved in cap-dependent protein translation, which when phosphorylated causes global translation attenuation. eIF2α phosphorylation is mediated by 4 kinases, which, together with their downstream signaling cascades, constitute the integrated stress response (ISR). While the ISR is activated by stresses commonly observed in neurodegeneration, such as oxidative stress, endoplasmic reticulum stress, and inflammation, it is a canonically adaptive signaling cascade. However, chronic activation of the ISR can contribute to neurodegenerative phenotypes such as neuronal death, memory impairments, and protein aggregation via apoptotic induction and other maladaptive outcomes downstream of phospho-eIF2α-mediated translation inhibition, including neuroinflammation and altered amyloidogenic processing, plausibly in a feed-forward manner. This review examines evidence that dysregulated eIF2a phosphorylation acts as a driver of neurodegeneration, including a survey of observations of ISR signaling in human disease, inspection of the overlap between ISR signaling and neurodegenerative phenomenon, and assessment of recent encouraging findings ameliorating neurodegeneration using developing pharmacological agents which target the ISR. In doing so, gaps in the field, including crosstalk of the ISR kinases and consideration of ISR signaling in nonneuronal central nervous system cell types, are highlighted.

Mounting evidence suggests that antiretroviral drugs may contribute to the persistence of HIV-associated neurocognitive disorders (HAND), which impact 30%–50% of HIV-infected patients in the post-antiretroviral era. We previously reported that two first generation HIV protease inhibitors, ritonavir and saquinavir, induced oxidative stress, with subsequent neuronal death in vitro, which was reversed by augmentation of the endogenous antioxidant response by monomethyl fumarate. We herein determined whether two newer-generation PIs, darunavir and lopinavir, were deleterious to neurons in vitro. Further, we expanded our assessment to include three integrase strand transfer inhibitors, raltegravir, dolutegravir, and elvitegravir. We found that only lopinavir and elvitegravir were neurotoxic to primary rat neuroglial cultures as determined by the loss of microtubule-associated protein 2 (MAP2). Intriguingly, lopinavir but not elvitegravir led to oxidative stress and induced the endogenous antioxidant response (EAR). Furthermore, neurotoxicity of lopinavir was blocked by pharmacological augmentation of the endogenous antioxidant heme oxygenase-1 (HO-1), expanding our previous finding that protease inhibitor-induced neurotoxicity was mediated by oxidative stress. Conversely, elvitegravir but not lopinavir led to increased eIF2α phosphorylation, indicating the activation of a common adaptive pathway termed the integrated stress response (ISR), and elvitegravir-mediated neurotoxicity was partially alleviated by the ISR inhibitor trans-ISRIB, suggesting ISR as a promoter of elvitegravir-associated neurotoxicity. Overall, we found that neurotoxicity was induced only by a subset of protease inhibitors and integrase strand transfer inhibitors, providing evidence for class- and drug-specific neurotoxic effects of antiretroviral drugs. Future in vivo studies will be critical to confirm the neurotoxicity profiles of these drugs for incorporation of these findings into patient management. The EAR and ISR pathways are potential access points for the development of adjunctive therapies to complement antiretroviral therapies and limit their contribution to HAND persistence.