Neuropathogenic role of astrocyte-derived extracellular vesicles in HIV-associated neurocognitive disorders.

Our previous findings demonstrated that astrocytic HIF-1α plays a major role in HIV-1 Tat-mediated amyloidosis which can lead to Alzheimer's-like pathology-a comorbidity of HIV-Associated Neurocognitive Disorders (HAND). These amyloids can be shuttled in extracellular vesicles, and we sought to assess whether HIV-1 Tat stimulated astrocyte-derived EVs (ADEVs) containing the toxic amyloids could result in neuronal injury in vitro and in vivo. We thus hypothesized that blocking HIF-1α could likely mitigate HIV-1 Tat-ADEV-mediated neuronal injury. Rat hippocampal neurons when exposed to HIV-1 Tat-ADEVs carrying the toxic amyloids exhibited amyloid accumulation and synaptodendritic injury, leading to functional loss as evidenced by alterations in miniature excitatory post synaptic currents. The silencing of astrocytic HIF-1α not only reduced the biogenesis of ADEVs, as well as amyloid cargos, but also ameliorated neuronal synaptodegeneration. Next, we determined the effect of HIV-1 Tat-ADEVs carrying amyloids in the hippocampus of naive mice brains. Naive mice receiving the HIV-1 Tat-ADEVs, exhibited behavioural changes, and Alzheimer's 's-like pathology accompanied by synaptodegeneration. This impairment(s) was not observed in mice injected with HIF-1α silenced ADEVs. This is the first report demonstrating the role of amyloid-carrying ADEVs in mediating synaptodegeneration leading to behavioural changes associated with HAND and highlights the protective role of HIF-1α.

Astrocytes & Astrocyte derived Extracellular Vesicles in Morphine Induced Amyloidopathy: Implications for Cognitive Deficits in Opiate Abusers.

While opiates like morphine play a major role in the pharmacotherapy for the control of pain associated with various diseases, paradoxically, their long-term use is associated with cognitive impairments. Furthermore, morphine administration has been shown to result in neuronal synaptodendritic injury in rodent brains, leading to neurodegeneration. We recently reported the role of astrocytes as contributors of amyloidosis associated with HIV-associated neurological disorders. Herein we hypothesize that morphine could induce astrocytic amyloidosis, which, in turn, could be disseminated to various regions in the brain by astrocyte-derived EVs (ADEVs). In this study we demonstrate brain region-specific up-regulation of astrocytic amyloids in morphine dependendent rhesus macaques. In addition, herein we also demonstrate increased expression of ß-site cleaving enzyme (BACE1), APP, and Aß in human primary astrocytes (HPAs) exposed to morphine. Mechanisms involved in this process included the up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the promoter of BACE1, leading to increased BACE1 activity and, generation of Aß 1-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1 mediated amyloidosis leading to astrocyte activation and neuroinflammation. We next sought to assess whether morphine-stimulated ADEVs could carry amyloid cargoes. Results showed that morphine exposure induced the release of morphine-ADEVs, carrying amyloids. Interestingly, silencing HIF-1α in astrocytes not only reduced the numbers of ADEV released, but also the packaging of amyloid cargos in the ADEVs. These findings were further validated in brain derived EVs (BEVs) isolated from macaques, wherein it was shown that BEVs from morphine-dependent macaques, carried varieties of amyloid cargoes including the cytokine IL-1ß. This is the first report implicating the role of HIF-1α-BACE1 axis in morphine-mediated induction of astrocytic amyloidosis, leading, in turn, to neuroinflammation and release of the amyloid cargoes via ADEVs. These findings set the groundwork for the future development of therapeutic strategies for targeting cognitive deficits in chronic opiate users.

HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis.

Increased life expectancy of patients diagnosed with HIV in the current era of antiretroviral therapy is unfortunately accompanied with the prevalence of HIV-associated neurocognitive disorders (HANDs) and risk of comorbidities such as Alzheimer-like pathology. HIV-1 transactivator of transcription (Tat) protein has been shown to induce the production of toxic neuronal amyloid protein and also enhance neurotoxicity. The contribution of astrocytes in Tat-mediated amyloidosis remains an enigma. We report here, in simian immunodeficiency virus (SIV)+ rhesus macaques and patients diagnosed with HIV, brain region-specific up-regulation of amyloid precursor protein (APP) and Aß (40 and 42) in astrocytes. In addition, we find increased expression of ß-site cleaving enzyme (BACE1), APP, and Aß in human primary astrocytes (HPAs) exposed to Tat. Mechanisms involved up-regulation of hypoxia-inducible factor (HIF-1α), its translocation and binding to the long noncoding RNA (lncRNA) BACE1-antisense transcript (BACE1-AS), resulting, in turn, in the formation of the BACE1-AS/BACE1 RNA complex, subsequently leading to increased BACE1 protein, and activity and generation of Aß-42. Gene silencing approaches confirmed the regulatory role of HIF-1α in BACE1-AS/BACE1 in Tat-mediated amyloidosis. This is the first report implicating the role of the HIF-1α/lncRNABACE1-AS/BACE1 axis in Tat-mediated induction of astrocytic amyloidosis, which could be targeted as adjunctive therapies for HAND-associated Alzheimer-like comorbidity.

NF-κB Duplications in the Promoter-Variant HIV-1C LTR Impact Inflammation Without Altering Viral Replication in the Context of Simian Human Immunodeficiency Viruses and Opioid-Exposure.

Recent spread of the promoter variant (4-κB) Human immunodeficiency virus-1 clade C (HIV-1C) strain is attributed to duplication of the Nuclear Factor Kappa B (NF-κB) binding sites and potential increased heroin consumption in India. To study the underlying biology of 4-κB HIV-1C in rhesus macaques, we engineered a promoter-chimera variant (4NF-κB) Simian Human Immunodeficiency Virus (SHIV) by substituting the HIV-1C Long Terminal Repeat (LTR) region consisting of 4 NF-κB and 3 Sp-1 sites with the corresponding segment in the LTR of SHIV AD8EO. The wild-type (3NF-κB) promoter-chimera SHIV was generated by inactivating the 5' proximal NF-κB binding site in SHIV 4NF-κB. CD8-depleted rhesus macaque PBMCs (RM-PBMCs) were infected with the promoter-chimera and AD8EO SHIVs to determine the effects of opioid-exposure on inflammation, NF-κB activation, neurotoxicity in neuronal cells and viral replication. Morphine-exposure of RM-PBMCs infected with SHIVs 4NF-κB, 3NF-κB, and AD8EO altered cellular transcript levels of monocyte chemoattractant protein 1, interleukin 6, interleukin 1ß, and Tumor Necrosis Factor α. Of note, divergent alteration of the cytokine transcript levels was observed with these promoter-chimera wild-type and variant SHIVs. NF-κB activation was observed during infection of all three SHIVs with morphine-exposure. Finally, we observed that SHIV AD8EO infection and exposure to both morphine and naloxone had the greatest impact on the neurotoxicity. The promoter-chimera SHIV 4NF-κB and SHIV 3NF-κB did not have a similar effect on neurotoxicity as compared to SHIV AD8EO. All SHIVs replicated efficiently at comparable levels in RM-PBMCs and morphine-exposure did not alter viral replication kinetics. Future in vivo studies in rhesus macaques will provide greater understanding of 4-κB HIV-1C viral immunopathogenesis and onset of disease in the central nervous system during morphine-exposure.

Strategies for the use of Extracellular Vesicles for the Delivery of Therapeutics.

Extracellular vesicles (EVs) are nanosized, membrane-bound vesicles released from eukaryotic and prokaryotic cells that can transport cargo containing DNA, RNA, lipids and proteins, between cells as a means of intercellular communication. Although EVs were initially considered to be cellular debris deprived of any essential biological functions, emerging literature highlights the critical roles of EVs in the context of intercellular signaling, maintenance of tissue homeostasis, modulation of immune responses, inflammation, cancer progression, angiogenesis, and coagulation under both physiological and pathological states. Based on the ability of EVs to shuttle proteins, lipids, carbohydrates, mRNAs, long non-coding RNAs (lncRNAs), microRNAs, chromosomal DNA, and mitochondrial DNA into target cells, the presence and content of EVs in biofluids have been exploited for biomarker research in the context of diagnosis, prognosis and treatment strategies. Additionally, owing to the characteristics of EVs such as stability in circulation, biocompatibility as well as low immunogenicity and toxicity, these vesicles have become attractive systems for the delivery of therapeutics. More recently, EVs are increasingly being exploited as conduits for delivery of therapeutics for anticancer strategies, immunomodulation, targeted drug delivery, tissue regeneration, and vaccination. In this review, we highlight and discuss the multiple strategies that are employed for the use of EVs as delivery vehicles for therapeutic agents, including the potential advantages and challenges involved. Graphical abstract.

PDGF/PDGFR axis in the neural systems.

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are expressed in several cell types including the brain cells such as neuronal progenitors, neurons, astrocytes, and oligodendrocytes. Emerging evidence shows that PDGF-mediated signaling regulates diverse functions in the central nervous system (CNS) such as neurogenesis, cell survival, synaptogenesis, modulation of ligand-gated ion channels, and development of specific types of neurons. Interestingly, PDGF/PDFGR signaling can elicit paradoxical roles in the CNS, depending on the cell type and the activation stimuli and is implicated in the pathogenesis of various neurodegenerative diseases. This review summarizes the role of PDGFs/PDGFRs in several neurodegenerative diseases such as Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, brain cancer, cerebral ischemia, HIV-1 and drug abuse. Understanding PDGF/PDGFR signaling may lead to novel approaches for the future development of therapeutic strategies for combating CNS pathologies.

Morphine-Mediated Brain Region-Specific Astrocytosis Involves the ER Stress-Autophagy Axis.

A recent study from our lab has revealed a link between morphine-mediated autophagy and synaptic impairment. The current study was aimed at investigating whether morphine-mediated activation of astrocytes involved the ER stress/autophagy axis. Our in vitro findings demonstrated upregulation of GFAP indicating astrocyte activation with a concomitant increase in the production of proinflammatory cytokines in morphine-exposed human astrocytes. Using both pharmacological and gene-silencing approaches, it was demonstrated that morphine-mediated defective autophagy involved upstream activation of ER stress with subsequent downstream astrocyte activation via the µ-opioid receptor (MOR). In vivo validation demonstrated preferential activation of ER stress/autophagy axis in the areas of the brain not associated with pain such as the basal ganglia, frontal cortex, occipital cortex, and the cerebellum of morphine-dependent rhesus macaques, and this correlated with increased astrocyte activation and neuroinflammation. Interventions aimed at blocking either the MOR or ER stress could thus likely be developed as promising therapeutic targets for abrogating morphine-mediated astrocytosis.

Impairment of blood brain barrier is related with the neuroinflammation induced peripheral immune status in intracerebroventricular colchicine injected rats: An experimental study with mannitol.

The neurodegeneration in AD patients may be associated with changes of peripheral immune responses. Some peripheral immune responses are altered due to neuroinflammation in colchicine induced AD (cAD) rats. The leaky blood brain barrier (BBB) in cAD-rats may be involved in inducing peripheral inflammation, though there is no report in this regard. Therefore, the present study was designed to investigate the role of BBB in cADrats by altering the BBB in a time dependent manner with injection (i.v.) of mannitol (BBB opener). The inflammatory markers in the brain and serum along with the peripheral immune responses were measured after 30 and 60min of mannitol injection in cAD rats. The results showed higher inflammatory markers in the hippocampus and serum along with alterations in peripheral immune parameters in cAD rats. Although the hippocampal inflammatory markers did not further change after mannitol injection in cAD rats, the serum inflammatory markers and peripheral immune responses were altered and these changes were greater after 60min than that of 30min of mannitol injection. The present study shows that the peripheral immune responses in cAD rats after 30 and 60min of mannitol injection are related to magnitude of impairment of BBB in these conditions. It can be concluded from this study that impairment of BBB in cAD rats is related to the changes of peripheral immune responses observed in that condition.

NMDA receptor is involved in neuroinflammation in intracerebroventricular colchicine-injected rats.

The neurodegeneration in intracerebroventricular (icv) colchicine injected (ICIR) rats is linked with neuroinflammation. Glutamate excitotoxicity through NMDA receptors is involved with the neuroinflammation in some animal models of Alzheimer Disease (AD), but it has not been explored in ICIR rats. The aim of this study was to investigate the role of NMDA receptors (by blocking it's activity with memantine) in colchicine-induced neuroinflammation and neurodegeneration and impacts on peripheral immune parameters in ICIR rats. Levels of inflammatory markers (IL-1ß, TNFα, ROS, nitrite) in the hippocampus and serum, histopathology of the hippocampus and select peripheral immune parameters were measured 14 and 21-days after icv colchicine injection in rats. These parameters were also measured in rats that received daily per os administration of memantine (20 mg/kg) in both study durations. Neuroinflammation in the hippocampus of ICIR rats was associated with neurodegeneration (chromatolysis, plaque formation), along with changes in inflammatory markers in the serum and alterations in peripheral immune parameters (phagocytic activity of WBC and splenic PMN, cytotoxic activity/leukocyte adhesion inhibition by splenic MNC). Administration of memantine to ICIR rats resulted in mitigation of colchicine-induced inflammation in the hippocampus, inflammatory markers in the serum and neurodegeneration and also led to recovery of the measured immune endpoints; most of these effects were greater with the longer duration of study. Phagocytic activity of WBC and splenic PMN cells appeared to correlate with levels of the measured central inflammatory markers. It appears from the results that neuroinflammation might be linked with the NMDA receptor activity in ICIR rats and that this receptor is involved in the process of progressive neuroinflammation and neurodegeneration in the hippocampus of ICIR and potentially in immunomodulation in these same hosts.

Protoporphyrin IX-induced structural and functional changes in human red blood cells, haemoglobin and myoglobin.

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin : protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.

Hematoporphyrin interacts with myoglobin and alters its functions.

The binding parameters of hematoporphyrin, a photosensitizing drug used in photodynamic therapy, interacting with myoglobin, an oxygen storage protein, have been studied spectrofluorometrically and spectrophotometrically. Two concentration ranges of hematoporphyrin, representing significantly monomeric and aggregated (dimeric) states have been used. The binding affinity constant (K) decreases and the possible number of binding sites (p) increases as the porphyrin changes from significantly monomeric state to predominantly dimeric state. Titration of the protein with hematoporphyrin in a spectrophotometric study (differential spectroscopy) exhibits an isosbestic point indicating a ground state complex formation. The interaction leads to a conformational change of the protein as observed in a circular dichroism study. The hematoporphyrin-myoglobin interaction causes oxygen release from the protein and it varies with the stoichiometric ratio of the porphyrin:protein. Hematoporphyrin also increases the myoglobin-catalysed hydrogen peroxide-mediated oxidation of o-dianisidine and NADH. These findings on the effects of hematoporphrin-myoglobin interaction should be given due consideration in therapeutic uses of the porphyrin and its derivatives.