A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders.

Astrocytes are one of the most numerous glial cells in the central nervous system (CNS) and provide essential support to neurons to ensure CNS health and function. During a neuropathological challenge, such as during human immunodeficiency virus (HIV)-1 infection or (METH)amphetamine exposure, astrocytes shift their neuroprotective functions and can become neurotoxic. Identifying cellular and molecular mechanisms underlying astrocyte dysfunction are of heightened importance to optimize the coupling between astrocytes and neurons and ensure neuronal fitness against CNS pathology, including HIV-1-associated neurocognitive disorders (HAND) and METH use disorder. Mitochondria are essential organelles for regulating metabolic, antioxidant, and inflammatory profiles. Moreover, endoplasmic reticulum (ER)-associated signaling pathways, such as calcium and the unfolded protein response (UPR), are important messengers for cellular fate and function, including inflammation and mitochondrial homeostasis. Increasing evidence supports that the three arms of the UPR are involved in the direct contact and communication between ER and mitochondria through mitochondria-associated ER membranes (MAMs). The current study investigated the effects of HIV-1 infection and chronic METH exposure on astrocyte ER and mitochondrial homeostasis and then examined the three UPR messengers as potential regulators of astrocyte mitochondrial dysfunction. Using primary human astrocytes infected with pseudotyped HIV-1 or exposed to low doses of METH for 7 days, astrocytes had increased mitochondrial oxygen consumption rate (OCR), cytosolic calcium flux and protein expression of UPR mediators. Notably, inositol-requiring protein 1α (IRE1α) was most prominently upregulated following both HIV-1 infection and chronic METH exposure. Moreover, pharmacological inhibition of the three UPR arms highlighted IRE1α as a key regulator of astrocyte metabolic function. To further explore the regulatory role of astrocyte IRE1α, astrocytes were transfected with an IRE1α overexpression vector followed by activation with the proinflammatory cytokine interleukin 1ß. Overall, our findings confirm IRE1α modulates astrocyte mitochondrial respiration, glycolytic function, morphological activation, inflammation, and glutamate uptake, highlighting a novel potential target for regulating astrocyte dysfunction. Finally, these findings suggest both canonical and non-canonical UPR mechanisms of astrocyte IRE1α. Thus, additional studies are needed to determine how to best balance astrocyte IRE1α functions to both promote astrocyte neuroprotective properties while preventing neurotoxic properties during CNS pathologies.

Astrocyte HIV-1 Tat Differentially Modulates Behavior and Brain MMP/TIMP Balance During Short and Prolonged Induction in Transgenic Mice.

Despite effective antiretroviral therapy (ART), mild forms of HIV-associated neurocognitive disorders (HAND) continue to afflict approximately half of all people living with HIV (PLWH). As PLWH age, HIV-associated inflammation perturbs the balance between brain matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs), likely contributing to neuropathogenesis. The MMP/TIMP balance is associated with cognition, learning, and memory, with TIMPs eliciting neuroprotective effects. Dysregulation of the MMP/TIMP balance was evident in the brains of PLWH where levels of TIMP-1, the inducible family member, were significantly lower than non-infected controls, and MMPs were elevated. Here, we evaluated the MMP/TIMP levels in the doxycycline (DOX)-induced glial fibrillary acidic protein promoter-driven HIV-1 transactivator of transcription (Tat) transgenic mouse model. The HIV-1 protein Tat is constitutively expressed by most infected cells, even during ART suppression of viral replication. Many studies have demonstrated indirect and direct mechanisms of short-term Tat-associated neurodegeneration, including gliosis, blood-brain barrier disruption, elevated inflammatory mediators and neurotoxicity. However, the effects of acute vs. prolonged exposure on Tat-induced dysregulation remain to be seen. This is especially relevant for TIMP-1 as expression was previously shown to be differentially regulated in human astrocytes during acute vs. chronic inflammation. In this context, acute Tat expression was induced with DOX intraperitoneal injections over 3 weeks, while DOX-containing diet was used to achieve long-term Tat expression over 6 months. First, a series of behavior tests evaluating arousal, ambulation, anxiety, and cognition was performed to examine impairments analogous to those observed in HAND. Next, gene expression of components of the MMP/TIMP axis and known HAND-relevant inflammatory mediators were assessed. Altered anxiety-like, motor and/or cognitive behaviors were observed in Tat-induced (iTat) mice. Gene expression of MMPs and TIMPs was altered depending on the duration of Tat expression, which was independent of the HIV-associated neuroinflammation typically implicated in MMP/TIMP regulation. Collectively, we infer that HIV-1 Tat-mediated dysregulation of MMP/TIMP axis and behavioral changes are dependent on duration of exposure. Further, prolonged Tat expression demonstrates a phenotype comparable to asymptomatic to mild HAND manifestation in patients.

ß-Catenin Regulates Wound Healing and IL-6 Expression in Activated Human Astrocytes.

Reactive astrogliosis is prominent in most neurodegenerative disorders and is often associated with neuroinflammation. The molecular mechanisms regulating astrocyte-linked neuropathogenesis during injury, aging and human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are not fully understood. In this study, we investigated the implications of the wingless type (Wnt)/ß-catenin signaling pathway in regulating astrocyte function during gliosis. First, we identified that HIV-associated inflammatory cytokines, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α induced mediators of the Wnt/ß-catenin pathway including ß-catenin and lymphoid enhancer-binding factor (LEF)-1 expression in astrocytes. Next, we investigated the regulatory role of ß-catenin on primary aspects of reactive astrogliosis, including proliferation, migration and proinflammatory responses, such as IL-6. Knockdown of ß-catenin impaired astrocyte proliferation and migration as shown by reduced cyclin-D1 levels, bromodeoxyuridine incorporation and wound healing. HIV-associated cytokines, IL-1ß alone and in combination with TNF-α, strongly induced the expression of proinflammatory cytokines including C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)8 and IL-6; however, only IL-6 levels were regulated by ß-catenin as demonstrated by knockdown and pharmacological stabilization. In this context, IL-6 levels were negatively regulated by ß-catenin. To better understand this relationship, we examined the crossroads between ß-catenin and nuclear factor (NF)-κB pathways. While NF-κB expression was significantly increased by IL-1ß and TNF-α, NF-κB levels were not affected by ß-catenin knockdown. IL-1ß treatment significantly increased glycogen synthase kinase (GSK)-3ß phosphorylation, which inhibits ß-catenin degradation. Further, pharmacological inhibition of GSK-3ß increased nuclear translocation of both ß-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. HIV+ human astrocytes show increased IL-6, ß-catenin and NF-κB expression levels and are interconnected by regulatory associations during HAND. In summary, our study demonstrates that HIV-associated inflammation increases ß-catenin pathway mediators to augment activated astrocyte responses including migration and proliferation, while mitigating IL-6 expression. These findings suggest that ß-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies, such as HAND.

Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.

Astrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1ß (IL-1ß) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1ß-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1ß-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1ß-ADEVs, as IL-1ß-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1ß-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.

IGF-IR determines the fates of BCR/ABL leukemia.

BACKGROUND: The tyrosine kinase receptor insulin-like growth factor 1 receptor (IGF-IR) contributes to the initiation and progression of many types of malignancies. We previously showed that IGF-2, which binds IGF-IR, is an extrinsic factor that supports the ex vivo expansion of hematopoietic stem cells (HSCs). We also demonstrated that IGF-IR is not required for HSC activity in vivo. METHODS AND RESULTS: Here we investigated the role of IGF-IR in chronic myeloid leukemia (CML) using the retroviral BCR/ABL transplantation mouse model. Existing antibodies against IGF-IR are not suitable for flow cytometry; therefore, we generated a fusion of the human IgG Fc fragment with mutant IGF-2 that can bind to IGF-IR. We used this fusion protein to evaluate mouse primary hematopoietic populations. Through transplantation assays with IGF-IR(+) and IGF-IR(-) cells, we demonstrated that IGF-IR is expressed on all mouse HSCs. The expression of IGF-IR is much higher on CML cells than on acute lymphoblastic leukemia (ALL) cells. The depletion of IGF-IR expression in BCR/ABL(+) cells led to the development of ALL (mostly T cell ALL) but not CML. Lack of IGF-IR resulted in decreased self-renewal of the BCR/ABL(+) CML cells in the serial replating assay. CONCLUSION: IGF-IR regulates the cell fate determination of BCR/ABL(+) leukemia cells and supports the self-renewal of CML cells.