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.

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.

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.

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.

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.

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.

Goldfish abducens motoneurons: physiological and anatomical specialization.

During natural movements, the motoneurons innervating a single muscle have different patterns of activity that are correlated with differences in synaptic input. The caudal abducens motoneurons fire phasically in synchronous bursts before rapid posterior eye movements; the rostral abducens motoneurons fire only tonically when the eye is fixed or moving slowly. This physiological difference is not related to motoneuron size. In this respect the abducens motoneurons violate the "size principle" that has been advanced for spinal motoneurons. The difference is probably related to the present finding that the caudal but not the rostral cells receive numerous electrical synapses that are known to have a role in synchronizing phasic activity.

Toward a functional architecture of the retina: serial reconstruction of adjacent ganglion cells.

Twenty adjacent ganglion cells in cat retina were partially reconstructed from electron micrographs of serial thin sections. Cells were classified by size and by dendritic branching patterns as alpha, beta, or gamma cells. The alpha and beta cells were further subdivided by differences in the laminar distribution of their dendrites in the inner plexiform layer. The distribution of synaptic contacts on the cells was distinctive for each of the five major classes. Contacts on the alpha and beta cells were mainly on the dendrites in the sublamina in which a cell's major dendritic arborization was contained.

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.

Evidence that vesicles on the synaptic ribbon of retinal bipolar neurons can be rapidly released.

We relate the ultrastructure of the giant bipolar synapse in goldfish retina to the jump in capacitance that accompanies depolarization-evoked exocytosis. Mean vesicle diameter is 29 +/- 4 nm, giving 26.4 aF/vesicle, so the maximum evoked capacitance (150 fF within 200 ms) represents fusion of about 5700 vesicles. Two terminals contained, respectively, 45 and 65 ribbon-type synaptic outputs, and a fully loaded ribbon tethers about 110 vesicles. Thus, the tethered pool, about 6000 vesicles, corresponds to the rapidly released pool. Further, the difference between small and large terminals in number of tethered vesicles matches their difference in capacitance jump. This suggests, within a "fire and reload" model of exocytosis, that the ribbon translocates synaptic vesicles very rapidly to membrane docking sites, supporting a maximum release rate of 500 vesicles/active zone/s, until the population of tethered vesicles is exhausted.

Mammalian rod terminal: architecture of a binary synapse.

The mammalian rod synapse transmits a binary signal (one photon or none) using tonic, rapid exocytosis. We constructed a quantitative, physical model of the synapse. Presynaptically, a single, linear active zone provides docking sites for approximately 130 vesicles, and a "ribbon" anchored to the active zone provides a depot for approximately 640 vesicles. Postsynaptically, 4 processes invaginate the terminal: 2 (known to have low affinity glutamate receptors) lie near the active zone (16 nm), and 2 (known to have high affinity glutamate receptors) lie at a distance (130-640 nm). The presynaptic structure seems designed to minimize fluctuations in tonic rate owing to empty docking sites, whereas the postsynaptic geometry may permit 1 vesicle to evoke an all-or-none response at all 4 postsynaptic processes.

Deciphering the retina's wiring diagram.

By simultaneously recording from retinal ganglion cells while stimulating a single cone, Chichilnisky and Baylor demonstrate that the strength of physiological connections within a retinal microcircuit is linearly proportional to the number of anatomically defined synapses.

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).

Localization of type I inositol 1,4,5-triphosphate receptor in the outer segments of mammalian cones.

Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and is extruded via a Na/Ca, K exchanger. We have identified another element in mammalian cones that might help to control cytoplasmic calcium. Reverse transcription-PCR performed on isolated photoreceptors identified mRNA for the SII- splice variant of the type I receptor for inositol 1,4,5-triphosphate (IP3), and Western blots showed that the protein also is expressed in outer segments. Immunocytochemistry showed type I IP3 receptor to be abundant in red-sensitive and green-sensitive cones of the trichromatic monkey retina, but it was negative or weakly expressed in blue-sensitive cones and rods. Similarly, the green-sensitive cones expressed the receptor in dichromatic retina (cat, rabbit, and rat), but the blue-sensitive cones did not. Immunostain was localized to disk and plasma membranes on the cytoplasmic face. To restore sensitivity after a light flash, cytoplasmic cGMP must rise to its basal level, and this requires cytoplasmic calcium to fall. Cessation of calcium release via the IP3 receptor might accelerate this fall and thus explain why the cone recovers much faster than the rod. Furthermore, because its own activity of the IP3 receptor depends partly on cytoplasmic calcium, the receptor might control the set point of cytoplasmic calcium and thus affect cone sensitivity.

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.

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.

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.