The Endolysosomal Transporter DMT1 is Required for Morphine Regulation of Neuronal Ferritin Heavy Chain.

NeuroHIV and other neurologic disorders present with altered iron metabolism in central nervous system neurons. Many people with HIV also use opioids, which can worsen neuroHIV symptoms by further dysregulating neuronal iron metabolism. Our previous work demonstrated that the µ-opioid agonist morphine causes neuronal endolysosomes to release their iron stores, and neurons respond by upregulating ferritin heavy chain (FHC), an iron storage protein associated with cognitive impairment in neuroHIV. Here, we investigated if this process required divalent metal transporter 1 (DMT1), a well-known iron transporter expressed on endolysosomes. We first optimized conditions to detect DMT1 isoforms (DMT1 1B ± iron responsive element) using fluorescently labeled rat DMT1 constructs expressed in HEK-293 cells. We also expressed these constructs in primary rat cortical neurons to compare their expression and subcellular distribution with endogenous DMT1 isoforms. We found endogenous DMT1 isoforms in the cytoplasm that colocalized with lysosomal-associated protein 1 (LAMP1), a marker of endolysosomes. Next, we blocked endogenous DMT1 isoforms using ebselen, a potent pharmacological inhibitor of DMT1 iron transport. Ebselen pre-treatment blocked morphine's ability to upregulate FHC protein, suggesting this pathway requires DMT1 iron transport from endolysosomes. This was further validated using viral-mediated genetic silencing of DMT1±IRE in cortical neurons, which also blocked FHC upregulation in the presence of morphine. Overall, our work demonstrates that the µ-opioid agonist morphine utilizes the endolysosomal iron transporter DMT1 to modulate neuronal cellular iron metabolism, upregulate FHC protein, and contribute to cognitive decline in neuroHIV. Morphine requires DMT1 to upregulate neuronal FHC. Cortical neurons treated with morphine release their endolysosomal iron stores to the cytoplasm and upregulate FHC, an iron storage protein associated with dendritic spine deficits and cognitive impairment in neuroHIV. This pathway requires the endolysosomal iron transporter DMT1, as pharmacological and genetic inhibitors of the transporter completely block morphine's ability to upregulate FHC. Created with BioRender.com .

The US9-Derived Protein gPTB9TM Modulates APP Processing Without Targeting Secretase Activities.

Alteration of neuronal protein processing is often associated with neurological disorders and is highly dependent on cellular protein trafficking. A prime example is the amyloidogenic processing of amyloid precursor protein (APP) in intracellular vesicles, which plays a key role in age-related cognitive impairment. Most approaches to correct this altered processing aim to limit enzymatic activities that lead to toxic products, such as protein cleavage by ß-secretase and the resulting amyloid ß production. A viable alternative is to direct APP to cellular compartments where non-amyloidogenic mechanisms are favored. To this end, we exploited the molecular properties of the herpes simplex virus 1 (HSV-1) transport protein US9 to guide APP interaction with preferred endogenous targets. Specifically, we generated a US9 chimeric construct that facilitates APP processing through the non-amyloidogenic pathway and tested it in primary cortical neurons. In addition to reducing amyloid ß production, our approach controls other APP-dependent biochemical steps that lead to neuronal deficits, including phosphorylation of APP and tau proteins. Notably, it also promotes the release of neuroprotective soluble αAPP. In contrast to other neuroprotective strategies, these US9-driven effects rely on the activity of endogenous neuronal proteins, which lends itself well to the study of fundamental mechanisms of APP processing/trafficking. Overall, this work introduces a new method to limit APP misprocessing and its cellular consequences without directly targeting secretase activity, offering a novel tool to reduce cognitive decline in pathologies such as Alzheimer's disease and HIV-associated neurocognitive disorders.

Opioid Modulation of Neuronal Iron and Potential Contributions to NeuroHIV.

Opioid use has substantially increased over recent years and remains a major driver of new HIV infections worldwide. Clinical studies indicate that opioids may exacerbate the symptoms of HIV-associated neurocognitive disorders (HAND), but the mechanisms underlying opioid-induced cognitive decline remain obscure. We recently reported that the µ-opioid agonist morphine increased neuronal iron levels and levels of ferritin proteins that store iron, suggesting that opioids modulate neuronal iron homeostasis. Additionally, increased iron and ferritin heavy chain protein were necessary for morphine's ability to reduce the density of thin and mushroom dendritic spines in cortical neurons, which are considered critical mediators of learning and memory, respectively. As altered iron homeostasis has been reported in HAND and related neurocognitive disorders like Alzheimer's, Parkinson's, and Huntington's disease, understanding how opioids regulate neuronal iron metabolism may help identify novel drug targets in HAND with potential relevance to these other neurocognitive disorders. Here, we review the known mechanisms of opioid-mediated regulation of neuronal iron and corresponding cellular responses and discuss the implications of these findings for patients with HAND. Furthermore, we discuss a new molecular approach that can be used to understand if opioid modulation of iron affects the expression and processing of amyloid precursor protein and the contributions of this pathway to HAND.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

The lipid raft-dwelling protein US9 can be manipulated to target APP compartmentalization, APP processing, and neurodegenerative disease pathogenesis.

The trafficking behavior of the lipid raft-dwelling US9 protein from Herpes Simplex Virus strikingly overlaps with that of the amyloid precursor protein (APP). Both US9 and APP processing machinery rely on their ability to shuttle between endosomes and plasma membranes, as well as on their lateral accumulation in lipid rafts. Therefore, repurposing US9 to track/modify these molecular events represents a valid approach to investigate pathological states including Alzheimer's disease and HIV-associated neurocognitive disorders where APP misprocessing to amyloid beta formation has been observed. Accordingly, we investigated the cellular localization of US9-driven cargo in neurons and created a US9-driven functional assay based on the exogenous enzymatic activity of Tobacco Etch Virus Protease. Our results demonstrate that US9 can direct and control cleavage of recombinant proteins exposed on the luminal leaflet of transport vesicles. Furthermore, we confirmed that US9 is associated with lipid-rafts and can target functional enzymes to membrane microdomains where pathologic APP-processing is thought to occur. Overall, our results suggest strongly that US9 can serve as a molecular driver that targets functional cargos to the APP machinery and can be used as a tool to study the contribution of lipid rafts to neurodegenerative disease conditions where amyloidogenesis has been implicated.

Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

Molecular features contributing to virus-independent intracellular localization and dynamic behavior of the herpesvirus transport protein US9.

Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining) this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts), defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36) have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

Regulation of neuronal P53 activity by CXCR 4.

Abnormal activation of CXCR 4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR 4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120(IIIB) or the endogenous CXCR 4 agonist, SDF-1 alpha. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR 4 by AMD 3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-alpha. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM 2 and p21. Conversely, SDF-1 alpha, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR 4 activation on neuronal survival in neuro-inflammatory disorders.

Apoptotic and antiapoptotic effects of CXCR4: is it a matter of intrinsic efficacy? Implications for HIV neuropathogenesis.

CXCR4, the specific receptor for the chemokine SDF-1 alpha that also binds CXCR4-using HIV gp120s, affects survival of different cell types, including neurons. However, current data show that the outcome of CXCR4 activation on neuronal survival may vary depending on the ligand and/or the cellular conditions. In this study, we have systematically compared the effects of SDF-1 alpha and gp120(IIIB) (with or without CD4) on several intracellular pathways involved in cell survival, including MAP kinases and Akt-dependent pathways. Our data show that gp120(IIIB) and SDF-1 alpha are both potent activators of MAP kinases in neuronal and non-neuronal cells, though the kinetic of these responses is slightly different. Furthermore, unlike SDF-1 alpha, and independently of CD4, gp120(IIIB) is unable to stimulate Akt and some of its antiapoptotic targets (NF-kappa B and MDM2)--despite its ability to activate other signaling pathways in the same conditions. Finally, the viral protein is more efficient in recruiting some effectors (e.g., JNK) than others in comparison with SDF-1 alpha (EC(50) = 0.1 vs. 0.6 nM). We conclude that the intrinsic efficacy of the two ligands is significantly different and is pathway dependent. These findings have important implications for our understanding of CXCR4-mediated responses in the CNS, as well as the role of this coreceptor in HIV neuropathogenesis.

Regulation of cell cycle proteins by chemokine receptors: A novel pathway in human immunodeficiency virus neuropathogenesis?

In order to test the hypothesis that alteration of cell cycle proteins are involved in the neuronal damage caused by human immunodeficiency virus (HIV), the authors have been studying the effect of chemokines on the CDK/Rb/E2F-1 pathway--which is involved in neuronal apoptosis and differentiation. First, they have asked whether CXCR4, the specific receptor for the chemokine SDF-1 and X4-using gp120s, can regulate Rb and E2F-1 activity in cultures of differentiated rat neurons. Although CCR3 and CCR5 are known to mediate infection of microglia by HIV-1, recent evidence indicate that CXCR4 also play important roles in HIV-induced neuronal injury, and dual-tropic isolates that use CXCR4 to infect macrophages have recently been reported. The authors have focused on two specific brain areas in which CXCR4 is physiologically relevant, i.e., the cerebellum and the hippocampus. So far, the data indicate that changes in the nuclear and cytosolic levels of Rb, which result in the functional loss of this protein, are associated with apoptosis in these neurons, and that SDF-1alpha and gp120IIIB affect this pathway. A summary of the findings are presented.

The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons.

Neurons express a variety of chemokine receptors that regulate neuronal signaling and survival, including CXCR4 and CCR5, the two major human immunodeficiency virus (HIV) coreceptors. However, the role of chemokine receptors in HIV neuropathology and neuroinflammatory disorders is still unclear. This study aims to determine whether chemokine receptors regulate the activity of cell-cycle proteins in neurons and evaluate the possibility that alterations of these proteins are involved in HIV neuropathogenesis. The authors studied the effect of the chemokine stromal cell-derived factor (SDF)-1alpha, the natural CXCR4 ligand, and an X4-using variant of gp120 on the activity of cell-cycle proteins involved in neuronal apoptosis and differentiation, such as Rb and E2F-1. Changes in expression, localization, and phosphorylation/activation of Rb and E2F-1 induced by SDF-1alpha (20 nM) gp120(IIIB) (200 pM) were analyzed in primary cultures of rat neurons and in a human cell line expressing recombinant CXCR4. The data indicate that changes in the nuclear and cytosolic levels of Rb--which result in the functional loss of this protein--are associated with apoptosis in hippocampal or cerebellar granule neurons and in cell lines. SDF-1alpha, which is able to rescue these neurons from apoptosis, induces a time-dependent increase of total Rb expression while decreasing the nuclear content of phosphorylated (Ser780/Ser795) Rb and the transcriptional activity of E2F-1. The HIV envelope protein gp120(IIIB) exerts opposite effects at the nuclear level. These data indicate that CXCR4 affects cell-cycle proteins in neurons and raise the possibility that chemokines may contribute to neuronal survival by repressing the activity of E2F-dependent apoptotic genes and maintaining neurons in a highly differentiated and quiescent state. This state may be altered during neuroinflammatory conditions and/or by HIV-derived proteins.