Time-dependent, HIV-Tat-induced perturbation of human neurons in vitro: towards a model for the molecular pathology of HIV-associated neurocognitive disorders

A significant proportion of human immunodeficiency virus type 1 (HIV)-positive individuals are affected by the cognitive, motor and behavioral dysfunction that characterizes HIV-associated neurocognitive disorders (HAND). While the molecular etiology of HAND remains largely uncharacterized, HIV transactivator of transcription (HIV-Tat) is thought to be an important etiological cause. Here we have used mass spectrometry (MS)-based discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time. We identified over 4000 protein groups (false discovery rate <0.01) in this system with 131, 118 and 45 protein groups differentially expressed at 6, 24 and 48 h post treatment, respectively. Alterations in the expression of proteins involved in gene expression and cytoskeletal maintenance were particularly evident. In tandem with proteomic evidence of cytoskeletal dysregulation we observed HIV-Tat induced functional alterations, including a reduction of neuronal intrinsic excitability as assessed by patch-clamp electrophysiology. Our findings may be relevant for understanding in vivo molecular mechanisms in HAND

Noninvasive optical inhibition with a red-shifted microbial rhodopsin

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.McGovern Institute for Brain Research at MIT (Razin Fellowship)United States. Defense Advanced Research Projects Agency. Living Foundries Program (HR0011-12-C-0068)Harvard-MIT Joint Research Grants Program in Basic NeuroscienceHuman Frontier Science Program (Strasbourg, France)Institution of Engineering and Technology (A. F. Harvey Prize)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) ProgramNew York Stem Cell Foundation (Robertson Investigator Award)National Institutes of Health (U.S.) (New Innovator Award 1DP2OD002002)National Institute of General Medical Sciences (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1RC1MH088182)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (Career Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS0848804)Society for Neuroscience (Research Award for Innovation in Neuroscience)Wallace H. Coulter FoundationNational Institutes of Health (U.S.) (RO1 MH091220-01)Whitehall FoundationEsther A. & Joseph Klingenstein Fund, Inc.JPB FoundationPIIF FundingNational Institute of Mental Health (U.S.) (R01-MH102441-01)National Institutes of Health (U.S.) (DP2-OD-017366-01)Massachusetts Institute of Technology. Simons Center for the Social Brai

Molecular analysis of hepatitis B virus (HBV) in an HIV co-infected patient with reactivation of occult HBV infection following discontinuation of lamivudine-including antiretroviral therapy

Abstract Background Occult hepatitis B virus (HBV) infection (OBI) is characterized by HBV DNA persistence even though the pattern of serological markers indicates an otherwise resolved HBV infection. Although OBI is usually clinically silent, immunocompromised patients may experience reactivation of the liver disease. Case presentation We report the case of an individual with human immunodeficiency virus (HIV) infection and anti-HBV core antibody positivity, who experienced severe HBV reactivation after discontinuation of lamivudine-including antiretroviral therapy (ART). HBV sequencing analysis showed a hepatitis B surface antigen escape mutant whose presence in an earlier sample excluded reinfection. Molecular sequencing showed some differences between two isolates collected at a 9-year interval, indicating HBV evolution. Resumption of ART containing an emtricitabine/tenofovir combination allowed control of plasma HBV DNA, which fell to undetectable levels. Conclusion This case stresses the ability of HBV to evolve continuously, even during occult infection, and the effectiveness of ART in controlling OBI reactivation in HIV-infected individuals.</p

Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice

BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need

Diapause as escape strategy to exposure to toxicants: response of Brachionus calyciforus to arsenic

Invertebrate organisms commonly respond to environmental fluctuation by entering diapause. Production of diapause in monogonont rotifers involves a previous switch from asexual to partial sexual reproduction. Although zooplankton have been used in ecotoxicological assays, often their true vulnerability to toxicants is underestimated by not incorporating the sexual phase. We experimentally analyzed traits involved in sexual reproduction and diapause in the cyclically parthenogenetic freshwater rotifer, Brachionus calyciflorus, exposed to arsenic, a metalloid naturally found in high concentrations in desert zones, focusing on the effectiveness of diapause as an escape response in the face of an adverse condition. Addition of sublethal concentrations of arsenic modified the pattern of diapause observed in the rotifer: investment in diapause with arsenic addition peaked earlier and higher than in non-toxicant conditions, which suggests that sexual investment could be enhanced in highly stressed environmental conditions by increased responsiveness to stimulation. Nevertheless, eggs produced in large amount with arsenic, were mostly low quality, and healthy-looking eggs had lower hatching success, therefore it is unclear whether this pattern is optimum in an environment with arsenic, or if rather arsenic presence in water bodies disturbs the optimal allocation of offspring entering diapause. We observed high accumulation of arsenic in organisms exposed to constant concentration after several generations, which suggests that arsenic may be accumulated transgenerationally. The sexual phase in rotifers may be more sensitive to environmental conditions than the asexual one, therefore diapause attributes should be considered in ecotoxicological assessment because of its ecological and evolutionary implications on lakes biodiversity

Spatial and temporal dynamics in the ionic driving force for GABA(A) receptors.

It is becoming increasingly apparent that the strength of GABAergic synaptic transmission is dynamic. One parameter that can establish differences in the actions of GABAergic synapses is the ionic driving force for the chloride-permeable GABA(A) receptor (GABA(A)R). Here we review some of the sophisticated ways in which this ionic driving force can vary within neuronal circuits. This driving force for GABA(A)Rs is subject to tight spatial control, with the distribution of Cl⁻ transporter proteins and channels generating regional variation in the strength of GABA(A)R signalling across a single neuron. GABA(A)R dynamics can result from short-term changes in their driving force, which involve the temporary accumulation or depletion of intracellular Cl⁻. In addition, activity-dependent changes in the expression and function of Cl⁻ regulating proteins can result in long-term shifts in the driving force for GABA(A)Rs. The multifaceted regulation of the ionic driving force for GABA(A)Rs has wide ranging implications for mature brain function, neural circuit development, and disease

Optogenetic silencing strategies differ in their effects on inhibitory synaptic transmission.

Optogenetic silencing using light-driven ion fluxes permits rapid and effective inhibition of neural activity. Using rodent hippocampal neurons, we found that silencing activity with a chloride pump can increase the probability of synaptically evoked spiking after photoactivation; this did not occur with a proton pump. This effect can be accounted for by changes to the GABA(A) receptor reversal potential and demonstrates an important difference between silencing strategies

Ion dynamics during seizures

Changes in membrane voltage brought about by ion fluxes through voltage and transmitter-gated channels represent the basis of neural activity. As such, electrochemical gradients across the membrane determine the direction and driving force for the flow of ions and are therefore crucial in setting the properties of synaptic transmission and signal propagation. Ion concentration gradients are established by a variety of mechanisms, including specialized transporter proteins. However, transmembrane gradients can be affected by ionic fluxes through channels during periods of elevated neural activity, which in turn are predicted to influence the properties of on-going synaptic transmission. Such activity-induced changes to ion concentration gradients are a feature of both physiological and pathological neural processes. An epileptic seizure is an example of severely perturbed neural activity, which is accompanied by pronounced changes in intracellular and extracellular ion concentrations. Appreciating the factors that contribute to these ion dynamics is critical if we are to understand how a seizure event evolves and is sustained and terminated by neural tissue. Indeed, this issue is of significant clinical importance as status epilepticus—a type of seizure that does not stop of its own accord—is a life-threatening medical emergency. In this review we explore how the transmembrane concentration gradient of the six major ions (K+, Na+, Cl−, Ca2+, H+and HCO−3) is altered during an epileptic seizure. We will first examine each ion individually, before describing how multiple interacting mechanisms between ions might contribute to concentration changes and whether these act to prolong or terminate epileptic activity. In doing so, we will consider how the availability of experimental techniques has both advanced and restricted our ability to study these phenomen

Chloride dynamics alter the input-output properties of neurons

Fast synaptic inhibition is a critical determinant of neuronal output, with subcellular targeting of synaptic inhibition able to exert different transformations of the neuronal input-output function. At the receptor level, synaptic inhibition is primarily mediated by chloride-permeable Type A GABA receptors. Consequently, dynamics in the neuronal chloride concentration can alter the functional properties of inhibitory synapses. How differences in the spatial targeting of inhibitory synapses interact with intracellular chloride dynamics to modulate the input-output function of neurons is not well understood. To address this, we developed computational models of multi-compartment neurons that incorporate experimentally parametrised mechanisms to account for neuronal chloride influx, diffusion, and extrusion. We found that synaptic input (either excitatory, inhibitory, or both) can lead to subcellular variations in chloride concentration, despite a uniform distribution of chloride extrusion mechanisms. Accounting for chloride changes resulted in substantial alterations in the neuronal input-output function. This was particularly the case for peripherally targeted dendritic inhibition where dynamic chloride compromised the ability of inhibition to offset neuronal input-output curves. Our simulations revealed that progressive changes in chloride concentration mean that the neuronal input-output function is not static but varies significantly as a function of the duration of synaptic drive. Finally, we found that the observed effects of dynamic chloride on neuronal output were mediated by changes in the dendritic reversal potential for GABA. Our findings provide a framework for understanding the computational effects of chloride dynamics on dendritically targeted synaptic inhibition