Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

Depressive-like Behavior Is Accompanied by Prefrontal Cortical Innate Immune Fatigue and Dendritic Spine Losses after HIV-1 Tat and Morphine Exposure.

Opioid use disorder (OUD) and HIV are comorbid epidemics that can increase depression. HIV and the viral protein Tat can directly induce neuronal injury within reward and emotionality brain circuitry, including the prefrontal cortex (PFC). Such damage involves both excitotoxic mechanisms and more indirect pathways through neuroinflammation, both of which can be worsened by opioid co-exposure. To assess whether excitotoxicity and/or neuroinflammation might drive depressive behaviors in persons infected with HIV (PWH) and those who use opioids, male mice were exposed to HIV-1 Tat for eight weeks, given escalating doses of morphine during the last two weeks, and assessed for depressive-like behavior. Tat expression decreased sucrose consumption and adaptability, whereas morphine administration increased chow consumption and exacerbated Tat-induced decreases in nesting and burrowing-activities associated with well-being. Across all treatment groups, depressive-like behavior correlated with increased proinflammatory cytokines in the PFC. Nevertheless, supporting the theory that innate immune responses adapt to chronic Tat exposure, most proinflammatory cytokines were unaffected by Tat or morphine. Further, Tat increased PFC levels of the anti-inflammatory cytokine IL-10, which were exacerbated by morphine administration. Tat, but not morphine, decreased dendritic spine density on layer V pyramidal neurons in the anterior cingulate. Together, our findings suggest that HIV-1 Tat and morphine differentially induce depressive-like behaviors associated with increased neuroinflammation, synaptic losses, and immune fatigue within the PFC.

Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia.

SUMMARY STATEMENT: HIV/HIV-1 Tat and morphine independently increase pathologic phosphorylation of TAR DNA binding protein 43 in the striatum. HIV- and opioid-induced pathologic phosphorylation of TAR DNA binding protein 43 may involve enhanced CK2 activity and protein levels.

Progressive Degeneration and Adaptive Excitability in Dopamine D1 and D2 Receptor-Expressing Striatal Neurons Exposed to HIV-1 Tat and Morphine.

The striatum is especially vulnerable to HIV-1 infection, with medium spiny neurons (MSNs) exhibiting marked synaptodendritic damage that can be exacerbated by opioid use disorder. Despite known structural defects in MSNs co-exposed to HIV-1 Tat and opioids, the pathophysiological sequelae of sustained HIV-1 exposure and acute comorbid effects of opioids on dopamine D1 and D2 receptor-expressing (D1 and D2) MSNs are unknown. To address this question, Drd1-tdTomato- or Drd2-eGFP-expressing reporter and conditional HIV-1 Tat transgenic mice were interbred. MSNs in ex vivo slices from male mice were assessed by whole-cell patch-clamp electrophysiology and filled with biocytin to explore the functional and structural effects of progressive Tat and acute morphine exposure. Although the excitability of both D1 and D2 MSNs increased following 48 h of Tat exposure, D1 MSN firing rates decreased below control (Tat-) levels following 2 weeks and 1 month of Tat exposure but returned to control levels after 2 months. D2 neurons continued to display Tat-dependent increases in excitability at 2 weeks, but also returned to control levels following 1 and 2 months of Tat induction. Acute morphine exposure increased D1 MSN excitability irrespective of the duration of Tat exposure, while D2 MSNs were variably affected. That D1 and D2 MSN excitability would return to control levels was unexpected since both subpopulations displayed significant synaptodendritic degeneration and pathologic phospho-tau-Thr205 accumulation following 2 months of Tat induction. Thus, despite frank morphologic damage, D1 and D2 MSNs uniquely adapt to sustained Tat and acute morphine insults.

Accelerated brain aging with opioid misuse and HIV: New insights on the role of glially derived pro-inflammation mediators and neuronal chloride homeostasis.

Opioid use disorder (OUD) has become a national crisis and contributes to the spread of human immunodeficiency virus (HIV) infection. Emerging evidence and advances in experimental models, methodology, and our understanding of disease processes at the molecular and cellular levels reveal that opioids per se can directly exacerbate the pathophysiology of neuroHIV. Despite substantial inroads, the impact of OUD on the severity, development, and prognosis of neuroHIV and HIV-associated neurocognitive disorders is not fully understood. In this review, we explore current evidence that OUD and neuroHIV interact to accelerate cognitive deficits and enhance the neurodegenerative changes typically seen with aging, through their effects on neuroinflammation. We suggest new thoughts on the processes that may underlie accelerated brain aging, including dysregulation of neuronal inhibition, and highlight findings suggesting that opioids, through actions at the µ-opioid receptor, interact with HIV in the central nervous system to promote unique structural and functional comorbid deficits not seen in either OUD or neuroHIV alone.

Independent actions by HIV-1 Tat and morphine to increase recruitment of monocyte-derived macrophages into the brain in a region-specific manner.

Despite advances in the treatment of human immunodeficiency virus (HIV), approximately one-half of people infected with HIV (PWH) experience neurocognitive impairment. Opioid use disorder (OUD) can exacerbate the cognitive and pathological changes seen in PWH. HIV increases inflammation and immune cell trafficking into the brain; however, less is known about how opioid use disorder affects the recruitment of immune cells. Accordingly, we examined the temporal consequences of HIV-1 Tat and/or morphine on the recruitment of endocytic cells (predominantly perivascular macrophages and microglia) in the dorsal striatum and hippocampus by infusing multi-colored, fluorescently labeled dextrans before and after exposure. To address this question, transgenic mice that conditionally expressed HIV-1 Tat (Tat+), or their control counterparts (Tat-), received three sequential intracerebroventricular (i.c.v.) infusions of Cascade Blue-, Alexa Fluor 488-, and Alexa Fluor 594-labeled dextrans, respectively infused 1 day before, 1-day after, or 13-days after morphine and/or Tat exposure. At the end of the study, the number of cells labeled with each fluorescent dextran were counted. The data demonstrated a significantly higher influx of newly-labeled cells into the perivascular space than into the parenchyma. In the striatum, Tat or morphine exposure increased the number of endocytic cells in the perivascular space, while only morphine increased the recruitment of endocytic cells into the parenchyma. In the hippocampus, morphine (but not Tat) increased the influx of dextran-labeled cells into the perivascular space, but there were too few labeled cells within the hippocampal parenchyma to analyze. Collectively, these data suggest that HIV-1 Tat and morphine act independently to increase the recruitment of endocytic cells into the brain in a region-specific manner.

Neurodegeneration Within the Amygdala Is Differentially Induced by Opioid and HIV-1 Tat Exposure.

Opioid use disorder (OUD) is a critical problem that contributes to the spread of HIV and may intrinsically worsen neuroHIV. Despite the advent of combined antiretroviral therapies (cART), about half of persons infected with HIV (PWH) experience cognitive and emotional deficits that can be exacerbated by opioid abuse. HIV-1 Tat is expressed in the central nervous system (CNS) of PWH on cART and is thought to contribute to neuroHIV. The amygdala regulates emotion and memories associated with fear and stress and is important in addiction behavior. Notwithstanding its importance in emotional saliency, the effects of HIV and opioids in the amygdala are underexplored. To assess Tat- and morphine-induced neuropathology within the amygdala, male Tat transgenic mice were exposed to Tat for 8 weeks and administered saline and/or escalating doses of morphine twice daily (s.c.) during the last 2 weeks of Tat exposure. Eight weeks of Tat exposure decreased the acoustic startle response and the dendritic spine density in the basolateral amygdala, but not the central nucleus of the amygdala. In contrast, repeated exposure to morphine alone, but not Tat, increased the acoustic startle response and whole amygdalar levels of amyloid-ß (Aß) monomers and oligomers and tau phosphorylation at Ser396, but not neurofilament light chain levels. Co-exposure to Tat and morphine decreased habituation and prepulse inhibition to the acoustic startle response and potentiated the morphine-induced increase in Aß monomers. Together, our findings indicate that sustained Tat and morphine exposure differentially promote synaptodendritic degeneration within the amygdala and alter sensorimotor processing.

Hippocampal blood-brain barrier of methamphetamine self-administering HIV-1 transgenic rats.

Combined antiretroviral therapy for HIV infection reduces plasma viral load and prolongs life. However, the brain is a viral reservoir, and pathologies such as cognitive decline and blood-brain barrier (BBB) disruption persist. Methamphetamine abuse is prevalent among HIV-infected individuals. Methamphetamine and HIV toxic proteins can disrupt the BBB, but it is unclear if there exists a common pathway by which HIV proteins and methamphetamine induce BBB damage. Also unknown are the BBB effects imposed by chronic exposure to HIV proteins in the comorbid context of chronic methamphetamine abuse. To evaluate these scenarios, we trained HIV-1 transgenic (Tg) and non-Tg rats to self-administer methamphetamine using a 21-day paradigm that produced an equivalency dose range at the low end of the amounts self-titrated by humans. Markers of BBB integrity were measured for the hippocampus, a brain region involved in cognitive function. Outcomes revealed that tight junction proteins, claudin-5 and occludin, were reduced in Tg rats independent of methamphetamine, and this co-occurred with increased levels of lipopolysaccharide, albumin (indicating barrier breakdown) and matrix metalloproteinase-9 (MMP-9; indicating barrier matrix disruption); reductions in GFAP (indicating astrocytic dysfunction); and microglial activation (indicating inflammation). Evaluations of markers for two signaling pathways that regulate MMP-9 transcription, NF-κB and ERK/∆FosB revealed an overall genotype effect for NF-κB. Methamphetamine did not alter measurements from Tg rats, but in non-Tg rats, methamphetamine reduced occludin and GFAP, and increased MMP-9 and NF-κB. Study outcomes suggest that BBB dysregulation resulting from chronic exposure to HIV-1 proteins or methamphetamine both involve NF-κB/MMP-9.

Morphine and HIV-1 Tat interact to cause region-specific hyperphosphorylation of tau in transgenic mice.

Opiate abuse is prevalent among HIV-infected individuals and may exacerbate HIV-associated age-related neurocognitive disorders. However, the extent to which HIV and opiates converge to accelerate pathological traits indicative of brain aging remains unknown. The pathological phospho-isotypes of tau (pSer396, pSer404, pThr205, pSer202, and pThr181) and the tau kinases GSK3ß and CDK5/p35 were explored in the striatum, hippocampus, and prefrontal cortex of inducible male and female HIV-1 Tat-transgenic mice, with some receiving escalating doses of morphine for 2 weeks. In the striatum of male mice, pSer396 was increased by co-exposure to morphine and Tat as compared to all other groups. Striatal pSer404 and pThr205 were increased by Tat alone, while pSer202 and pThr181 were unchanged. A comparison between Tat-transgenic female and male mice revealed disparate outcomes for pThr205. No other sex-related changes to tau phosphorylation were observed. In the hippocampus, Tat increased pSer396, while other phosphorylation sites were unchanged and pSer202 was not detected. In the prefrontal cortex, morphine increased pSer396 levels, which were unaffected by Tat, while other phosphorylation sites were unaffected. Assessment of tau kinases revealed no changes to striatal GSK3ß (phosphorylated or total) or the total CDK5 levels. Striatal levels of phosphorylated CDK5 and p35, the activator of CDK5, were increased by Tat and with morphine co-exposure, respectively. P35 levels positively correlated with those of pSer396 with Tat and morphine co-exposure. The results reveal region-specific hyperphosphorylation of tau induced by exposure to morphine, Tat, and unique morphine and Tat interactions.

Region-specific changes in markers of neuroplasticity revealed in HIV-1 transgenic rats by low-dose methamphetamine.

Methamphetamine abuse co-occurring with HIV infection presents neuropathology in brain regions that mediate reward and motivation. A neuronal signaling cascade altered acutely by meth and some HIV-1 proteins is the mitogen-activated protein kinase (MAPK) pathway. It remains unknown if chronic co-exposure to meth and HIV-1 proteins converge on MAPK in vivo. To make this determination, we studied young adult Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats that self-administered meth (0.02-0.04 mg/kg/0.05 ml iv infusion, 2 h/day for 21 days) and their saline-yoked controls. One day following the operant task, rats were killed. Brain regions involved in reward-motivation [i.e., nucleus accumbens (NA) and ventral pallidum (VP)], were assayed for a MAPK cascade protein, extracellular signal-regulated kinase (ERK), and a downstream transcription factor, ΔFosB. In the NA, activated (phosphorylated; p) ERK-to-ERK ratio (pERK/ERK) was increased in meth-exposed Tg rats versus saline Tg controls, and versus meth non-Tg rats. ΔFosB was increased in meth Tg rats versus saline and meth non-Tg rats. Assessment of two targets of ΔFosB-regulated transcription revealed (1) increased dopamine D1 receptor (D1R) immunoreactivity in the NA shell of Tg-meth rats versus saline Tg controls, but (2) no changes in the AMPA receptor subunit, GluA2. No changes related to genotype or meth occurred for ERK, ΔFosB or D1R protein in the VP. Results reveal a region-specific activation of ERK, and increases in ΔFosB and D1R expression induced by HIV-1 proteins and meth. Such effects may contribute to the neuronal and behavioral pathology associated with meth/HIV comorbidity.

Colon dysregulation in methamphetamine self-administering HIV-1 transgenic rats.

The integrity and function of the gut is impaired in HIV-infected individuals, and gut pathogenesis may play a role in several HIV-associated disorders. Methamphetamine is a popular illicit drug abused by HIV-infected individuals. However, the effect of methamphetamine on the gut and its potential to exacerbate HIV-associated gut pathology is not known. To shed light on this scenario, we evaluated colon barrier pathology in a rat model of the human comorbid condition. Intestinal barrier integrity and permeability were assessed in drug-naïve Fischer 344 HIV-1 transgenic (Tg) and non-Tg rats, and in Tg and non-Tg rats instrumented with jugular cannulae trained to self-administer methamphetamine or serving as saline-yoked controls. Intestinal permeability was determined by measuring the urine content of orally gavaged sugars. Intestinal barrier integrity was evaluated by immunoblotting or immunofluorescence of colon claudin-1 and zonula occludens-1 (ZO-1), two major tight junction proteins that regulate gut epithelial paracellular permeability. Both non-Tg and Tg rats self-administered moderate amounts of methamphetamine. These amounts were sufficient to increase colon permeability, reduce protein level of claudin-1, and reduce claudin-1 and ZO-1 immunofluorescence in Tg rats relative to non-Tg rats. Methamphetamine decreased tight junction immunofluorescence in non-Tg rats, with a similar, but non-significant trend observed in Tg rats. However, the effect of methamphetamine on tight junction proteins was subthreshold to gut leakiness. These findings reveal that both HIV-1 proteins and methamphetamine alter colon barrier integrity, and indicate that the gut may be a pathogenic site for these insults.