HIV-1 gp120 protein promotes HAND through the calcineurin pathway activation.

For over two decades, highly active antiretroviral therapy (HAART) was able to help prolong the life expectancy of people living with HIV-1 (PLWH) and eliminate the virus to an undetectable level. However, an increased prevalence of HIV- associated neurocognitive disorders (HAND) was observed. These symptoms range from neuronal dysfunction to cell death. Among the markers of neuronal deregulation, we cite the alteration of synaptic plasticity and neuronal communications. Clinically, these dysfunctions led to neurocognitive disorders such as learning alteration and loss of spatial memory, which promote premature brain aging even in HAART-treated patients. In support of these observations, we showed that the gp120 protein deregulates miR-499-5p and its downstream target, the calcineurin (CaN) protein. The gp120 protein also promotes the accumulation of calcium (Ca2+) and reactive oxygen species (ROS) inside the neurons leading to the activation of CaN and the inhibition of miR-499-5p. gp120 protein also caused mitochondrial fragmentation and changes in shape and size. The use of mimic miR-499 restored mitochondrial functions, appearance, and size. These results demonstrated the additional effect of the gp120 protein on neurons through the miR-499-5p/calcineurin pathway.

Disruption of Mitochondrial-associated ER membranes by HIV-1 tat protein contributes to premature brain aging.

INTRODUCTION: Mitochondrial-associated ER membranes (MAMs) control many cellular functions, including calcium and lipid exchange, intracellular trafficking, and mitochondrial biogenesis. The disruption of these functions contributes to neurocognitive disorders, such as spatial memory impairment and premature brain aging. Using neuronal cells, we demonstrated that HIV-1 Tat protein deregulates the mitochondria. METHODS& RESULTS: To determine the mechanisms, we used a neuronal cell line and showed that Tat-induced changes in expression and interactions of both MAM-associated proteins and MAM tethering proteins. The addition of HIV-1 Tat protein alters expression levels of PTPIP51 and VAPB proteins in the MAM fraction but not the whole cell. Phosphorylation of PTPIP51 protein regulates its subcellular localization and function. We demonstrated that the Tat protein promotes PTPIP51 phosphorylation on tyrosine residues and prevents its binding to VAPB. Treatment of the cells with a kinase inhibitor restores the PTPIP51-VAPB interaction and overcomes the effect of Tat. CONCLUSION: These results suggest that Tat disrupts the MAM, through the induction of PTPIP51 phosphorylation, leading to ROS accumulation, mitochondrial stress, and altered movement. Hence, we concluded that interfering in the MAM-associated cellular pathways contributes to spatial memory impairment and premature brain aging often observed in HIV-1-infected patients.

SARS-CoV-2 Causes Lung Inflammation through Metabolic Reprogramming and RAGE.

Clinical studies indicate that patients infected with SARS-CoV-2 develop hyperinflammation, which correlates with increased mortality. The SARS-CoV-2/COVID-19-dependent inflammation is thought to occur via increased cytokine production and hyperactivity of RAGE in several cell types, a phenomenon observed for other disorders and diseases. Metabolic reprogramming has been shown to contribute to inflammation and is considered a hallmark of cancer, neurodegenerative diseases, and viral infections. Malfunctioning glycolysis, which normally aims to convert glucose into pyruvate, leads to the accumulation of advanced glycation end products (AGEs). Being aberrantly generated, AGEs then bind to their receptor, RAGE, and activate several pro-inflammatory genes, such as IL-1b and IL-6, thus, increasing hypoxia and inducing senescence. Using the lung epithelial cell (BEAS-2B) line, we demonstrated that SARS-CoV-2 proteins reprogram the cellular metabolism and increase pyruvate kinase muscle isoform 2 (PKM2). This deregulation promotes the accumulation of AGEs and senescence induction. We showed the ability of the PKM2 stabilizer, Tepp-46, to reverse the observed glycolysis changes/alterations and restore this essential metabolic process.

HIV-1 gp120 Impairs Spatial Memory Through Cyclic AMP Response Element-Binding Protein.

HIV-associated neurocognitive disorders (HAND) remain an unsolved problem that persists despite using antiretroviral therapy. We have obtained data showing that HIV-gp120 protein contributes to neurodegeneration through metabolic reprogramming. This led to decreased ATP levels, lower mitochondrial DNA copy numbers, and loss of mitochondria cristae, all-important for mitochondrial biogenesis. gp120 protein also disrupted mitochondrial movement and synaptic plasticity. Searching for the mechanisms involved, we found that gp120 alters the cyclic AMP response element-binding protein (CREB) phosphorylation on serine residue 133 necessary for its function as a transcription factor. Since CREB regulates the promoters of PGC1α and BDNF genes, we found that CREB dephosphorylation causes PGC1α and BDNF loss of functions. The data was validated in vitro and in vivo. The negative effect of gp120 was alleviated in cells and animals in the presence of rolipram, an inhibitor of phosphodiesterase protein 4 (PDE4), restoring CREB phosphorylation. We concluded that HIV-gp120 protein contributes to HAND via inhibition of CREB protein function.

Metabolic Reprogramming in HIV-Associated Neurocognitive Disorders.

A significant number of patients infected with HIV-1 suffer from HIV-associated neurocognitive disorders (HAND) such as spatial memory impairments and learning disabilities (SMI-LD). SMI-LD is also observed in patients using combination antiretroviral therapy (cART). Our lab has demonstrated that the HIV-1 protein, gp120, promotes SMI-LD by altering mitochondrial functions and energy production. We have investigated cellular processes upstream of the mitochondrial functions and discovered that gp120 causes metabolic reprogramming. Effectively, the addition of gp120 protein to neuronal cells disrupted the glycolysis pathway at the pyruvate level. Looking for the players involved, we found that gp120 promotes increased expression of polypyrimidine tract binding protein 1 (PTBP1), causing the splicing of pyruvate kinase M (PKM) into PKM1 and PKM2. We have also shown that these events lead to the accumulation of advanced glycation end products (AGEs) and prevent the cleavage of pro-brain-derived neurotrophic factor (pro-BDNF) protein into mature brain-derived neurotrophic factor (BDNF). The accumulation of proBDNF results in signaling that increases the expression of the inducible cAMP early repressor (ICER) protein which then occupies the cAMP response element (CRE)-binding sites within the BDNF promoters II and IV, thus altering normal synaptic plasticity. We reversed these events by adding Tepp-46, which stabilizes the tetrameric form of PKM2. Therefore, we concluded that gp120 reprograms cellular metabolism, causing changes linked to disrupted memory in HIV-infected patients and that preventing the disruption of the metabolism presents a potential cure against HAND progression.

Hallmarks of Metabolic Reprogramming and Their Role in Viral Pathogenesis.

Metabolic reprogramming is a hallmark of cancer and has proven to be critical in viral infections. Metabolic reprogramming provides the cell with energy and biomass for large-scale biosynthesis. Based on studies of the cellular changes that contribute to metabolic reprogramming, seven main hallmarks can be identified: (1) increased glycolysis and lactic acid, (2) increased glutaminolysis, (3) increased pentose phosphate pathway, (4) mitochondrial changes, (5) increased lipid metabolism, (6) changes in amino acid metabolism, and (7) changes in other biosynthetic and bioenergetic pathways. Viruses depend on metabolic reprogramming to increase biomass to fuel viral genome replication and production of new virions. Viruses take advantage of the non-metabolic effects of metabolic reprogramming, creating an anti-apoptotic environment and evading the immune system. Other non-metabolic effects can negatively affect cellular function. Understanding the role metabolic reprogramming plays in viral pathogenesis may provide better therapeutic targets for antivirals.

HIV-1 Vpr protein impairs lysosome clearance causing SNCA/alpha-synuclein accumulation in neurons.

Despite the promising therapeutic effects of combinatory antiretroviral therapy (cART), 20% to 30% of HIV/AIDS patients living with long term infection still exhibit related cognitive and motor disorders. Clinical studies in HIV-infected patients revealed evidence of basal ganglia dysfunction, tremors, fine motor movement deficits, gait, balance, and increased risk of falls. Among older HIV+ adults, the frequency of cases with SNCA/α-synuclein staining is higher than in older healthy persons and may predict an increased risk of developing a neurodegenerative disease. The accumulation of SNCA aggregates known as Lewy Bodies is widely described to be directly linked to motor dysfunction. These aggregates are naturally removed by Macroautophagy/autophagy, a cellular housekeeping mechanism, that can be disturbed by HIV-1. The molecular mechanisms involved in linking HIV-1 proteins and autophagy remain mostly unclear and necessitates further exploration. We showed that HIV-1 Vpr protein triggers the accumulation of SNCA in neurons after decreasing lysosomal acidification, deregulating lysosome positioning, and the expression levels of several proteins involved in lysosomal maturation. Viruses and retroviruses such as HIV-1 are known to manipulate autophagy in order to use it for their replication while blocking the degradative final step, which could destroy the virus itself. Our study highlights how the suppression of neuronal autophagy by HIV-1 Vpr is a mechanism leading to toxic protein aggregation and neurodegeneration.Abbreviations: BLOC1: Biogenesis of Lysosome-related Organelles Complex 1; CART: combinatory antiretroviral therapy; CVB: coxsackievirus; DAPI: 4',6-diamidino-2-phenylindole; DENV: dengue virus; GFP: green fluorescent protein; HCV: hepatitis C virus; HCMV: human cytomegalovirus; HIV: human immunodeficiency virus; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; VSV: Indiana vesiculovirus; LTR: Long Terminal Repeat; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MLBs: multilamellar bodies; RIPA: Radioimmunoprecipitation assay buffer; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tat: transactivator of TAR; TEM: transmission electron microscope; Vpr: Viral protein R.

Why do SARS-CoV-2 NSPs rush to the ER?

SARS-CoV-2, which led to the 2020 global pandemic, is responsible for the Coronavirus Disease 2019 (COVID-19), a respiratory illness, and presents a tropism for the central nervous system. Like most members of this family, the virus is composed of structural and non-structural proteins (NSPs). The non-structural proteins are critical elements of the replication and transcription complex (RTC), as well as immune system evasion. Through hijacking the endoplasmic reticulum (ER) membrane, NSPs help the virus establish the RTC, inducing ER stress after membrane rearrangement and causing severe neuronal disturbance. In this review, we focus on the role of Nsp3, 4, and 6 in intracellular membrane rearrangement and evaluate the potential disruption of the central nervous system and the neurodegeneration which it could trigger. Studies of these NSPs will not only bring to light their specific role in viral infection but also facilitate the discovery of novel targeted drugs.

Safety learning system development--incident reporting component for family practice.

OBJECTIVE: To determine the required components for developing the reporting components of a safety learning system (SLS) for community-based family practice. METHODS: Multiple databases were searched for all languages for all types of papers related to medical safety in community practice: Books@Ovid, BIOSIS Previews, CDSR, ACP Journal Club, DARE, CCTR, Ageline, AMED, CINAHL, EMBASE, HealthSTAR, Ovid MEDLINE In-Process, Other Non-Indexed Citations, Ovid MEDLINE, PsycINFO, HAPI and PsycBOOKS. A grey literature search was done in Google. RESULTS: The online search identified 190 papers. English abstracts were read and the full papers (or chapters) were retrieved for 90, of which 18 were deemed appropriate. The grey literature search revealed 18 additional papers, and an additional 12 papers were identified from bibliographies of included papers. The common themes identified from the articles became the main consideration for developing an SLS for family practice and include current and past initiatives, system design, incident reporting form and classification system. CONCLUSION: There is a small but growing body of literature concerning the requirements for developing the reporting component of an SLS for family practice. For the reporting component of an SLS to be successful, there needs to be strong leadership, voluntary reporting, legal protection and feedback to reporters.

Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons.

In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic "hot spots." Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. Electron microscopy (EM) analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles approximately 30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway.

Synaptic ribbon. Conveyor belt or safety belt?

The synaptic ribbon in neurons that release transmitter via graded potentials has been considered as a conveyor belt that actively moves vesicles toward their release sites. But evidence has accumulated to the contrary, and it now seems plausible that the ribbon serves instead as a safety belt to tether vesicles stably in mutual contact and thus facilitate multivesicular release by compound exocytosis.

Cellular basis for the response to second-order motion cues in Y retinal ganglion cells.

We perceive motion when presented with spatiotemporal changes in contrast (second-order cue). This requires linear signals to be rectified and then summed in temporal order to compute direction. Although both operations have been attributed to cortex, rectification might occur in retina, prior to the ganglion cell. Here we show that the Y ganglion cell does indeed respond to spatiotemporal contrast modulations of a second-order motion stimulus. Responses in an OFF ganglion cell are caused by an EPSP/IPSP sequence evoked from within the dendritic field; in ON cells inhibition is indirect. Inhibitory effects, which are blocked by tetrodotoxin, clamp the response near resting potential thus preventing saturation. Apparently the computation for second-order motion can be initiated by Y cells and completed by cortical cells that sum outputs of multiple Y cells in a directionally selective manner.

Microcircuits for night vision in mouse retina.

Because the mouse retina has become an important model system, we have begun to identify its specific neuron types and their synaptic connections. Here, based on electron micrographs of serial sections, we report that the wild-type mouse retina expresses the standard rod pathways known in other mammals: (1) rod --> cone (via gap junctions) to inject rod signals into the cone bipolar circuit; and (2) rod --> rod bipolar --> AII amacrine --> cone bipolar --> ganglion cell. The mouse also expresses another rod circuit: a bipolar cell with cone input also receives rod input at symmetrical contacts that express ionotropic glutamate receptors (Hack et al., 1999, 2001). We show that this rod-cone bipolar cell sends an axon to the outer (OFF) strata of the inner plexiform layer to form ribbon synapses with ganglion and amacrine cells. This rod-cone bipolar cell receives direct contacts from only 20% of all rod terminals. However, we also found that rod terminals form gap junctions with each other and thus establish partial syncytia that could pool rod signals for direct chemical transmission to the OFF bipolar cell. This third rod pathway probably explains the rod responses that persist in OFF ganglion cells after the well known rod pathways are blocked (Soucy et al., 1998).

Bipolar cells contribute to nonlinear spatial summation in the brisk-transient (Y) ganglion cell in mammalian retina.

The receptive field of the Y-ganglion cell comprises two excitatory mechanisms: one integrates linearly over a narrow field, and the other integrates nonlinearly over a wide field. The linear mechanism has been attributed to input from bipolar cells, and the nonlinear mechanism has been attributed to input from a class of amacrine cells whose nonlinear "subunits" extend across the linear receptive field and beyond. However, the central component of the nonlinear mechanism could in theory be driven by bipolar input if that input were rectified. Recording intracellularly from the Y-cell in guinea pig retina, we blocked the peripheral component of the nonlinear mechanism with tetrodotoxin and found the remaining nonlinear receptive field to be precisely co-spatial with the central component of the linear receptive field. Both linear and nonlinear mechanisms were caused by an excitatory postsynaptic potential that reversed near 0 mV. The nonlinear mechanism depended neither on acetylcholine nor on feedback involving GABA or glycine. Thus the central components of the ganglion cell's linear and nonlinear mechanisms are apparently driven by synapses from the same rectifying bipolar cell.

The light response of ON bipolar neurons requires G[alpha]o.

ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G(o) might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the alpha subunit of G(o) (Galpha(o)-/-). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod- and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the Galpha(o) null mouse establishes that the light response of the ON bipolar cell requires G(o). This represents the first function to be defined in vivo for the alpha subunit of the most abundant G-protein of the brain.

Evidence that different cation chloride cotransporters in retinal neurons allow opposite responses to GABA.

GABA gating an anion channel primarily permeable to chloride can hyperpolarize or depolarize, depending on whether the chloride equilibrium potential (E(Cl)) is negative or positive, respectively, to the resting membrane potential (E(rest)). If the transmembrane Cl(-) gradient is set by active transport, those neurons or neuronal regions that exhibit opposite responses to GABA should express different chloride transporters. To test this, we immunostained retina for the K-Cl cotransporter (KCC2) that normally extrudes chloride and for the Na-K-Cl cotransporter (NKCC) that normally accumulates chloride. KCC2 was expressed wherever E(Cl) is either known or predicted to be negative to E(rest) (ganglion cells, bipolar axons, and OFF bipolar dendrites), whereas NKCC was expressed wherever E(Cl) is either known or predicted to be positive to E(rest) (horizontal cells and ON bipolar dendrites). Thus, in the retina, the opposite effects of GABA on different cell types and on different cellular regions are probably primarily determined by the differential targeting of these two chloride transporters.

Localization of mGluR6 to dendrites of ON bipolar cells in primate retina.

We prepared antibodies selective for the C-terminus of the human mGluR6 receptor and used confocal and electron microscopy to study the patterns of immunostaining in retina of monkey, cat, and rabbit. In all three species punctate stain was restricted to the outer plexiform layer. In monkey, stain was always observed in the central element of the postsynaptic "triad" of rod and cone terminals. In monkey peripheral retina, stain was seen only in central elements, but in the fovea, stain was also observed in some dendrites contacting the base of the cone terminal. S-cone terminals, identified by staining for S opsin, showed staining of postsynaptic dendrites. These were identified as dendrites of the ON S-cone bipolar cell by immunostaining for the marker cholecystokinin precursor. The staining pattern suggests that all types of ON bipolar cells, despite their marked differences in function, express a single isoform of mGluR6. Ultrastructurally, mGluR6 was located not on the tip of the central element, near the site of vesicle release, but on its base at the mouth of the invagination, 400-800 nm from the release site. Thus, the mGluR6 receptors of ON bipolar cells lie at about the same distance from sites of vesicle release as the iGluR receptors of OFF bipolar cells at the basal contacts.

Cost of cone coupling to trichromacy in primate fovea.

Cone synaptic terminals couple electrically to their neighbors. This reduces the amplitude of temporally uncorrelated voltage differences between neighbors. For an achromatic stimulus coarser than the cone mosaic, the uncorrelated voltage difference between neighbors represents mostly noise; so noise is reduced more than the signal. Here coupling improves signal-to-noise ratio and enhances contrast sensitivity. But for a chromatic stimulus the uncorrelated voltage difference between neighbors of different spectral type represents mostly signal; so signal would be reduced more than the noise. This cost of cone coupling to encoding chromatic signals was evaluated using a compartmental model of the foveal cone array. When cones sensitive to middle (M) and long (L) wavelengths alternated regularly, and the conductance between a cone and all of its immediate neighbors was 1,000 pS (approximately 2 connexons/cone pair), coupling reduced the difference between the L and M action spectra by nearly fivefold, from about 38% to 8%. However, L and M cones distribute randomly in the mosaic, forming small patches of like type, and within a patch the responses to a chromatic stimulus are correlated. In such a mosaic, coupling still reduced the difference between the L and M action spectra, but only by 2.4-fold, to about 18%. This result is independent of the L/M ratio. Thus "patchiness" of the L/M mosaic allows cone coupling to improve achromatic contrast sensitivity while minimizing the cost to chromatic sensitivity.

Functional circuitry of the retinal ganglion cell's nonlinear receptive field.

A retinal ganglion cell commonly expresses two spatially overlapping receptive field mechanisms. One is the familiar "center/surround," which sums excitation and inhibition across a region somewhat broader than the ganglion cell's dendritic field. This mechanism responds to a drifting grating by modulating firing at the drift frequency (linear response). Less familiar is the "nonlinear" mechanism, which sums the rectified output of many small subunits that extend for millimeters beyond the dendritic field. This mechanism responds to a contrast-reversing grating by modulating firing at twice the reversal frequency (nonlinear response). We investigated this nonlinear mechanism by presenting visual stimuli to the intact guinea pig retina in vitro while recording intracellularly from large brisk and sluggish ganglion cells. A contrast-reversing grating modulated the membrane potential (in addition to the firing rate) at twice the reversal frequency. This response was initially hyperpolarizing for some cells (either ON or OFF center) and initially depolarizing for others. Experiments in which responses to bars were summed in-phase or out-of-phase suggested that the single class of bipolar cells (either ON or OFF) that drives the center/surround response also drives the nonlinear response. Consistent with this, nonlinear responses persisted in OFF ganglion cells when ON bipolar cell responses were blocked by L-AP-4. Nonlinear responses evoked from millimeters beyond the ganglion cell were eliminated by tetrodotoxin. Thus, to relay the response from distant regions of the receptive field requires a spiking interneuron. Nonlinear responses from different regions of the receptive field added linearly.