Neurodegenerative Disorders of Alzheimer, Parkinsonism, Amyotrophic Lateral Sclerosis and Multiple Sclerosis: An Early Diagnostic Approach for Precision Treatment.

Neurodegenerative diseases (NDs) are characterised by progressive dysfunction of synapses, neurons, glial cells and their networks. Neurodegenerative diseases can be classified according to primary clinical features (e.g., dementia, parkinsonism, or motor neuron disease), anatomic distribution of neurodegeneration (e.g., frontotemporal degenerations, extrapyramidal disorders, or spinocerebellar degenerations), or principal molecular abnormalities. The most common neurodegenerative disorders are amyloidosis, tauopathies, a-synucleinopathy, and TAR DNA-binding protein 43 (TDP-43) proteopathy. The protein abnormalities in these disorders have abnormal conformational properties along with altered cellular mechanisms, and they exhibit motor deficit, mitochondrial malfunction, dysfunctions in autophagic-lysosomal pathways, synaptic toxicity, and more emerging mechanisms such as the roles of stress granule pathways and liquid-phase transitions. Finally, for each ND, microglial cells have been reported to be implicated in neurodegeneration, in particular, because the microglial responses can shift from neuroprotective to a deleterious role. Growing experimental evidence suggests that abnormal protein conformers act as seed material for oligomerization, spreading from cell to cell through anatomically connected neuronal pathways, which may in part explain the specific anatomical patterns observed in brain autopsy sample. In this review, we mention the human pathology of select neurodegenerative disorders, focusing on how neurodegenerative disorders (i.e., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis) represent a great healthcare problem worldwide and are becoming prevalent because of the increasing aged population. Despite many studies have focused on their etiopathology, the exact cause of these diseases is still largely unknown and until now with the only available option of symptomatic treatments. In this review, we aim to report the systematic and clinically correlated potential biomarker candidates. Although future studies are necessary for their use in early detection and progression in humans affected by NDs, the promising results obtained by several groups leads us to this idea that biomarkers could be used to design a potential therapeutic approach and preclinical clinical trials for the treatments of NDs.

Allosteric Modulation of Adenosine A2A Receptors as a New Therapeutic Avenue.

The therapeutic potential of targeting adenosine A2A receptors (A2ARs) is immense due to their broad expression in the body and central nervous system. The role of A2ARs in cardiovascular function, inflammation, sleep/wake behaviors, cognition, and other primary nervous system functions has been extensively studied. Numerous A2AR agonist and antagonist molecules are reported, many of which are currently in clinical trials or have already been approved for treatment. Allosteric modulators can selectively elicit a physiologic response only where and when the orthosteric ligand is released, which reduces the risk of an adverse effect resulting from A2AR activation. Thus, these allosteric modulators have a potential therapeutic advantage over classical agonist and antagonist molecules. This review focuses on the recent developments regarding allosteric A2AR modulation, which is a promising area for future pharmaceutical research because the list of existing allosteric A2AR modulators and their physiologic effects is still short.

Understanding preanalytical variables and their effects on clinical biomarkers of oncology and immunotherapy.

Identifying a suitable course of immunotherapy treatment for a given patient as well as monitoring treatment response is heavily reliant on biomarkers detected and quantified in blood and tissue biospecimens. Suboptimal or variable biospecimen collection, processing, and storage practices have the potential to alter clinically relevant biomarkers, including those used in cancer immunotherapy. In the present review, we summarize effects reported for immunologically relevant biomarkers and highlight preanalytical factors associated with specific analytical platforms and assays used to predict and gauge immunotherapy response. Given that many of the effects introduced by preanalytical variability are gene-, transcript-, and protein-specific, biospecimen practices should be standardized and validated for each biomarker and assay to ensure accurate results and facilitate clinical implementation of newly identified immunotherapy approaches.

Nanoproteomic analysis of ischemia-dependent changes in signaling protein phosphorylation in colorectal normal and cancer tissue.

BACKGROUND: Clinical diagnostic research relies upon the collection of tissue samples, and for those samples to be representative of the in vivo situation. Tissue collection procedures, including post-operative ischemia, can impact the molecular profile of the tissue at the genetic and proteomic level. Understanding the influence of factors such as ischemia on tissue samples is imperative in order to develop both markers of tissue quality and ultimately accurate diagnostic tests. METHODS: Using NanoPro1000 technology, a rapid and highly sensitive immunoassay platform, the phosphorylation status of clinically relevant cancer-related biomarkers in response to ischemia was quantified in tissue samples from 20 patients with primary colorectal cancer. Tumor tissue and adjacent normal tissue samples were collected and subjected to cold ischemia prior to nanoproteomic analysis of AKT, ERK1/2, MEK1/2, and c-MET. Ischemia-induced relative changes in overall phosphorylation and phosphorylation of individual isoforms were calculated and statistical significance determined. Any differences in baseline levels of phosphorylation between tumor tissue and normal tissue were also analyzed. RESULTS: Changes in overall phosphorylation of the selected proteins in response to ischemia revealed minor variations in both normal and tumor tissue; however, significant changes were identified in the phosphorylation of individual isoforms. In normal tissue post-operative ischemia, phosphorylation was increased in two AKT isoforms, two ERK1/2 isoforms, and one MEK1/2 isoform and decreased in one MEK1/2 isoform and one c-MET isoform. Following ischemia in tumor tissue, one AKT isoform showed decreased phosphorylation and there was an overall increase in unphosphorylated ERK1/2, whereas an increase in the phosphorylation of two MEK1/2 isoforms was observed. There were no changes in c-MET phosphorylation in tumor tissue. CONCLUSION: This study provides insight into the influence of post-operative ischemia on tissue sample biology, which may inform the future development of markers of tissue quality and assist in the development of diagnostic tests.

Inventing a co-axial atomic resolution patch clamp to study a single resonating protein complex and ultra-low power communication deep inside a living neuron cell.

To read the signals of single molecules in vitro on a surface, or inside a living cell or organ, we introduce a coaxial atom tip (coat) and a coaxial atomic patch clamp (COAPAP). The metal-insulator-metal cavity of these probes extends to the atomic scale (0.1[Formula: see text]nm), it eliminates the cellular or environmental noise with a S/N ratio 105. Five ac signals are simultaneously applied during a measurement by COAT and COAPAP to shield a true signal under environmental noise in five unique ways. The electromagnetic drive in the triaxial atomic tips is specifically designed to sense anharmonic vibrational and transmission signals for any system between 0.1[Formula: see text]nm and 50[Formula: see text]nm where the smallest nanopatch clamp cannot reach. COAT and COAPAP reliably pick up the atomic scale vibrations under the extreme noise of a living cell. Each protein's distinct electromagnetic, mechanical, electrical and ionic vibrational signature studied in vitro in a protected environment is found to match with the ones studied inside a live neuron. Thus, we could confirm that by using our probe blindly we could hold on to a single molecule or its complex in the invisible domain of a living cell. Our decade long investigations on perfecting the tools to measure bio-resonance of all forms and simultaneously in all frequency domains are summarized. It shows that the ratio of emission to absorption resonance frequencies of a biomaterial is around [Formula: see text], only a few in the entire em spectrum are active that regulates all other resonances, like mechanical, ionic, etc.

Inventing atomic resolution scanning dielectric microscopy to see a single protein complex operation live at resonance in a neuron without touching or adulterating the cell.

A substantial ion flow in a normally wet protein masks any other forms of signal transmission. We use hysteresis and linear conduction (both are artifacts) as a marker to precisely wet a protein, which restricts the ionic conduction (hysteresis disappears), and at the same time, it is not denatured (quantized conductance and Raman spectra are intact). Pure electric visualization of proteins at work by eliminating the screening of ions, electrons, would change the way we study biology. Here we discuss the technical challenges resolved for imaging a protein or live cell using nonlinear dielectric response (spatial distribution of conductance, capacitance and phase, GCP trio). We electromagnetically triggered electrical, mechanical, thermal and ionic resonant vibrations in a protein. During resonant oscillations, we imaged the protein using resonant scanning tunneling microscopy of biomaterials (Brestum) and during ionic firing we imaged live what happens inside an axon core of a neuron by using our atomic scale scanning dielectric microscopy (Asadim). Both Asadim and Brestum are housed in a homebuilt scanning tunneling microscope (bio-STM) and a special micro-grid developed by us (patent JP-5187804) for fractal supercomputing. We found the trick to turn a membrane transparent and see inside without making any physical contact. We image live that a protein molecule adopts a unique configuration for each resonance frequency, - thus far unknown to biology. "Membrane alone fires" is found to be wrong after a century, micro-neuro-filaments communicate prior to firing to decide its necessity and then regulate it suitably. We introduce a series of technologies e.g., fractal grid, point contact, micro THz antenna, to discover that from atomic structure to a living cell, the biomaterials vibrate collectively.

HIV-1 Tat neurotoxicity: a model of acute and chronic exposure, and neuroprotection by gene delivery of antioxidant enzymes.

HIV-associated neurocognitive disorder (HAND) is an increasingly common, progressive disease characterized by neuronal loss and progressively deteriorating CNS function. HIV-1 gene products, particularly gp120 and Tat elicit reactive oxygen species (ROS) that lead to oxidant injury and cause neuron apoptosis. Understanding of, and developing therapies for, HAND requires accessible models of the disease. We have devised experimental approaches to studying the acute and chronic effects of Tat on the CNS. We studied acute exposure by injecting recombinant Tat protein into the caudate-putamen (CP). Ongoing Tat expression, which more closely mimics HIV-1 infection of the brain, was studied by delivering Tat-expression over time using an SV40-derived gene delivery vector, SV(Tat). Both acute and chronic Tat exposure induced lipid peroxidation and neuronal apoptosis. Finally, prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/zinc superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), protected from Tat-induced apoptosis and oxidative injury. Thus, injection of recombinant HIV-1 Tat and the expression vector, SV(Tat), into the rat CP cause respectively acute or ongoing apoptosis and oxidative stress in neurons and may represent useful animal models for studying the pathogenesis and, potentially, treatment of HIV-1 Tat-related damage.

Role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage.

Chemokines may play a role in leukocyte migration across the blood-brain barrier (BBB) during neuroinflammation and other neuropathological processes, such as epilepsy. We investigated the role of the chemokine receptor CCR5 in seizures. We used a rat model based on intraperitoneal kainic acid (KA) administration. Four months before KA injection, adult rats were given femoral intramarrow inoculations of SV (RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) against CCR5, plus a marker epitope (AU1), or its monofunctional RNAi-carrying homologue, SV(RNAiR5). This treatment lowered expression of CCR5 in circulating cells. In control rats, seizures induced elevated expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, increased BBB leakage and CCR5(+) cells, as well as neuronal loss, inflammation, and gliosis in the hippocampi. Animals given either the bifunctional or the monofunctional vector were largely protected from KA-induced seizures, neuroinflammation, BBB damage, and neuron loss. Brain CCR5 mRNA was reduced. Rats receiving RNAiR5-bearing vectors showed far greater repair responses: increased neuronal proliferation, and decreased production of MIP-1α and RANTES. Controls received unrelated SV(BUGT) vectors. Decrease in CCR5 in circulating cells strongly protected from excitotoxin-induced seizures, BBB leakage, CNS injury, and inflammation, and facilitated neurogenic repair.

Biodegradable and Electrically Conductive Melanin-Poly (3-Hydroxybutyrate) 3D Fibrous Scaffolds for Neural Tissue Engineering Applications.

Due to the severity of peripheral nerve injuries (PNI) and spinal cord injuries (SCI), treatment options for patients are limited. In this context, biomaterials designed to promote regeneration and reinstate the lost function are being explored. Such biomaterials should be able to mimic the biological, chemical, and physical cues of the extracellular matrix for maximum effectiveness as therapeutic agents. Development of biomaterials with desirable physical, chemical, and electrical properties, however, has proven challenging. Here a novel biomaterial formulation achieved by blending the pigment melanin and the natural polymer Poly-3-hydroxybutyrate (PHB) is proposed. Physio-chemical measurements of electrospun fibers reveal a feature rich surface nano-topography, a semiconducting-nature, and brain-tissue-like poroviscoelastic properties. Resulting fibers improve cell adhesion and growth of mouse sensory and motor neurons, without any observable toxicity. Further, the presence of polar functional groups positively affect the kinetics of fibers degradation at a pH (≈7.4) comparable to that of body fluids. Thus, melanin-PHB blended scaffolds are found to be physio-chemically, electrically, and biologically compatible with neural tissues and could be used as a regenerative modality for neural tissue injuries. A biomaterial for scaffolds intended to promote regeneration of nerve tissue after injury is developed. This biomaterial, obtained by mixing the pigment melanin and the natural polymer PHB, is biodegradable, electrically conductive, and beneficial to the growth of motor and sensory neurons. Thus, it is believed that this biomaterial can be used in the context of healthcare applications.

Use of Microfluidics Chambers to Image Axonal transport in Adult Sensory Neurons.

Transport of cargoes along axons is crucial for ensuring effective neuronal function and survival. Lysosomes, which are membrane-bound organelles responsible for the degradation of macromolecules, are among the many cargoes being transported. Compartmentalized systems that allow for the separation of the somatic compartment from the axonal network, are widely used in the field of neurobiology and in the study of axonal transport in particular. Among the various solutions available, microfluidics chambers that take advantage of fluidic separation between different compartments, have seen widespread adoption. Said chambers are made of polydimethylsiloxane (PDMS), a transparent, gas permeable compound, which is compatible with fluorescence microscopy, and have significantly positively impacted cellular neuroscience, drastically increasing our understanding of axonal peripheral signaling. Here we describe a two-layered microfluidics chamber, engineered to allow for the culture of adult sensory neurons. This device was designed to promote the proper placement of adult sensory neurons in the somatic chamber in proximity of the microgrooves. We detail the production of the master mold, how to fabricate and assemble the device and how to disaggregate and load the cells in it. In addition, we provide details on how to conduct and analyze an axonal transport experiment using a custom made script in MATLAB designed by our laboratory.

HIV-1 gp120 upregulates matrix metalloproteinases and their inhibitors in a rat model of HIV encephalopathy.

Matrix metalloproteinases (MMPs) are implicated in diverse processes, such as neuroinflammation, leakiness of the blood-brain barrier (BBB) and direct cellular damage in neurodegenerative and other CNS diseases. Tissue destruction by MMPs is regulated by their endogenous tissue inhibitors (TIMPs). TIMPs prevent excessive MMP-related degradation of extracellular matrix components. In a rat model of human immunodeficiency virus (HIV)-related encephalopathy, we described MMP-2 and MMP-9 upregulation by HIV-1 envelope gp120, probably via gp120-induced reactive oxygen species. Antioxidant gene delivery blunted gp120-induced MMP production. We also studied the effect of gp120 on TIMP-1 and TIMP-2 production. TIMP-1 and TIMP-2 levels increased 6 h after gp120 injection into rat caudate-putamen (CP). TIMP-1 and TIMP-2 colocalized mainly with neurons (92 and 95%, respectively). By 24 h, expression of these protease inhibitors diverged, as TIMP-1 levels remained high but TIMP-2 subsided. Gene delivery of the antioxidant enzymes Cu/Zn superoxide dismutase or glutathione peroxidase into the CP before injecting gp120 there reduced levels of gp120-induced TIMP-1 and TIMP-2, recapitulating the effect of antioxidant enzymes on gp120-induced MMP-2 and MMP-9. A significant correlation was observed between MMP/TIMP upregulation and BBB leakiness. Thus, HIV-1 gp120 upregulated TIMP-1 and TIMP-2 in the CP. Prior antioxidant enzyme treatment mitigated production of these TIMPs, probably by reducing MMP expression.

Development of 3D culture scaffolds for directional neuronal growth using 2-photon lithography.

Conventional applications of transplant technology, applied to severe traumatic injuries of the nervous system, have met limited success in the clinics due to the complexity of restoring function to the damaged tissue. Neural tissue engineering aims to deploy scaffolds mimicking the physiological properties of the extracellular matrix to facilitate the elongation of axons and the repair of damaged nerves. However, the fabrication of ideal scaffolds with precisely controlled thickness, texture, porosity, alignment, and with the required mechanical strength, features needed for effective clinical applications, remains technically challenging. We took advantage of state-of-the-art 2-photon photolithography to fabricate highly ordered and biocompatible 3D nanogrid structures to enhance neuronal directional growth. First, we characterized the physical and chemical properties and proved the biocompatibility of said scaffolds by successfully culturing primary sensory and motor neurons on their surface. Interestingly, axons extended along the fibers with a high degree of alignment to the pattern of the nanogrid, as opposed to the lack of directionality observed on flat glass or polymeric surfaces, and could grow in 3D between different layers of the scaffold. The axonal growth pattern observed is highly desirable for the treatment of traumatic nerve damage occurring during peripheral and spinal cord injuries. Thus, our findings provide a proof of concept and explore the possibility of deploying aligned fibrous 3D scaffold/implants for the directed growth of axons, and could be used in the design of scaffolds targeted towards the restoration and repair of lost neuronal connections.

Viroinformatics-Based Analysis of SARS-CoV-2 Core Proteins for Potential Therapeutic Targets.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus for which no known effective antiviral drugs are available. In the present study, to accelerate the discovery of potential drug candidates, bioinformatics-based in silico drug discovery approaches are utilized. We performed multiple sequence alignments of the Spike (S) protein with 75 sequences of different viruses from the Orthocoronavirinae subfamily. This provided us with insights into the evolutionarily conserved domains that can be targeted using drugs or specific antibodies. Further, we analyzed the mechanism of SARS-CoV-2 core proteins, i.e., S and RdRp (RNA-dependent RNA polymerase), to elucidate how the virus infection can utilize hemoglobin to decrease the blood oxygen level. Moreover, after a comprehensive literature survey, more than 60 antiviral drugs were chosen. The candidate drugs were then ranked based on their potential to interact with the Spike and RdRp proteins of SARS-CoV-2. The present multidimensional study further advances our understanding of the novel viral molecular targets and potential of computational approaches for therapeutic assessments. The present study can be a steppingstone in the selection of potential drug candidates to be used either as a treatment or as a reference point when designing a new drug/antibody/inhibitory peptide/vaccine against SARS-CoV-2.

Sepsis Following Liver Biopsy in a Liver Transplant Recipient: Case Report and Review of Literature.

Percutaneous liver biopsy is a relatively safe procedure with low complication rates. Infections following liver biopsy are uncommon and can lead to a poor outcome. There are limited data on liver biopsy-related infections among liver transplant (LT) recipients. Also, there is a paucity of data regarding the use of prophylactic antibiotics in LT patients undergoing percutaneous liver biopsy. We report a case of systemic sepsis following percutaneous liver biopsy in a LT recipient with choledochojejunal anastomosis. This was followed by severe rejection and deterioration of liver function and recurrence of primary sclerosing cholangitis (PSC) to the extent that he has been listed for retransplantation. This case report emphasizes the potential risk of sepsis in LT recipients with bilioenteric anastomosis undergoing percutaneous liver biopsy. This increased risk may warrant periprocedural broad spectrum antibiotic prophylaxis, in this subgroup of patients.

Targeting the IL-2 inducible kinase in melanoma; a phase 2 study of ibrutinib in systemic treatment-refractory distant metastatic cutaneous melanoma: preclinical rationale, biology, and clinical activity (NCI9922).

BACKGROUND: IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. METHODS: We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). RESULTS: Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. CONCLUSION: Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.

Blood-brain barrier abnormalities caused by exposure to HIV-1 gp120--protection by gene delivery of antioxidant enzymes.

HIV-1 effects on the blood-brain barrier (BBB) structure and function are still poorly understood in animal models based on direct administration of recombinant HIV proteins. We therefore injected HIV-1 envelope glycoprotein, gp120, into rat caudate-putamens (CPs) and examined vascular integrity and function. Gp120 coimmunostained with endothelial cell marker, CD31. It induced apoptosis of endothelial cells in vitro and in vivo. BBB function was assessed by administering Evans Blue (EB) intravenously before injecting gp120. EB leaked near the site of gp120 administration. Within 1h after intra-CP gp120 injection, structures positive for endothelial markers ICAM-1 and RECA-1 were greatly decreased. Vascular density assessed by laminin immunostaining remained decreased 1 month after gp120 injection. RECA-1-positive cells expressed hydroxynonenal, a marker of lipid peroxidation and rSV40-mediated gene delivery of antioxidant enzymes protected the BBB from gp120-related injury. Extravasated IgG accumulated following intra-CP SV(gp120) injection, an experimental model of continuing gp120 exposure. Thus: acute and chronic exposure to gp120 disrupts the BBB; gp120-mediated BBB abnormalities are related to lesions of brain microvessels; and gp120 is directly toxic to brain endothelial cells.

Therapeutic potential of serotonin 4 receptor for chronic depression and its associated comorbidity in the gut.

The latest estimates from world health organization suggest that more than 450 million people are suffering from depression and other psychiatric conditions. Of these, 50-60% have been reported to have progression of gut diseases. In the last two decades, researchers introduced incipient physiological roles for serotonin (5-HT) receptors (5-HTRs), suggesting their importance as a potential pharmacological target in various psychiatric and gut diseases. A growing body of evidence suggests that 5-HT systems affect the brain-gut axis in depressive patients, which leads to gut comorbidity. Recently, preclinical trials of 5-HT4R agonists and antagonists were promising as antipsychotic and prokinetic agents. In the current review, we address the possible pharmacological role and contribution of 5-HT4R in the pathophysiology of chronic depression and associated gut abnormalities. Physiologically, during depression episodes, centers of the sympathetic and parasympathetic nervous system couple together with neuroendocrine systems to alter the function of hypothalamic-pituitary-adrenal (HPA) axis and enteric nervous system (ENS), which in turn leads to onset of gastrointestinal tract (GIT) disorders. Consecutively, the ENS governs a broad spectrum of physiological activities of gut, such as visceral pain and motility. During the stages of emotional stress, hyperactivity of the HPA axis alters the ENS response to physiological and noxious stimuli. Consecutively, stress-induced flare, swelling, hyperalgesia and altered reflexes in gut eventually lead to GIT disorders. In summary, the current review provides prospective information about the role and mechanism of 5-HT4R-based therapeutics for the treatment of depressive disorder and possible consequences for the gut via brain-gut axis interactions. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

A rat model of human immunodeficiency virus 1 encephalopathy using envelope glycoprotein gp120 expression delivered by SV40 vectors.

Human immunodeficiency virus 1 (HIV-1) encephalopathy is thought to result in part from the toxicity of HIV-1 envelope glycoprotein gp120 for neurons. Experimental systems for studying the effects of gp120 and other HIV proteins on the brain have been limited to the acute effects of recombinant proteins in vitro or in vivo in simian immunodeficiency virus-infected monkeys. We describe an experimental rodent model of ongoing gp120-induced neurotoxicity in which HIV-1 envelope is expressed in the brain using an SV40-derived gene delivery vector, SV(gp120). When it is inoculated stereotaxically into the rat caudate putamen, SV(gp120) caused a partly hemorrhagic lesion in which neuron and other cell apoptosis continues for at least 12 weeks. Human immunodeficiency virus gp120 is expressed throughout this time, and some apoptotic cells are gp120 positive. Malondialdehyde and 4-hydroxynonenal assays indicated that there was lipid peroxidation in these lesions. Prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/ zinc superoxide dismutase or glutathione peroxidase, was protective against SV(gp120)-induced oxidative injury and apoptosis. Thus, in vivo inoculation of SV(gp120) into the rat caudate putamen causes ongoing oxidative stress and apoptosis in neurons and may therefore represent a useful animal model for studying the pathogenesis and treatment of HIV-1 envelope-related brain damage.

HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: protection by rSV40 delivery of antioxidant enzymes.

Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.

CCR5Delta32 59537-G/A promoter polymorphism is associated with low translational efficiency and the loss of CCR5Delta32 protective effects.

We have recently demonstrated that the CCR5Delta32 protein interacts with CCR5 and CXCR4 and down-modulates their cell surface expression. We have also reported the absence of detectable expression of the truncated CCR5Delta32 protein in four out of six human immunodeficiency virus-infected (HIV(+)) CCR5(-/-) individuals. To explain the defect in protein expression in these samples, we cloned and sequenced the promoter regions of the six HIV(+) individuals. We have identified several polymorphisms in the CCR5Delta32 promoter region, but these polymorphisms were not associated with significant differences in mRNA levels. Coupled in vitro transcription/translation and polyribosome analysis demonstrated a strong association between a variant genotype designated CCR5Delta32 59537-A/A and a low translation efficiency. Protein analysis indicated that the peripheral blood mononuclear cells from two of the HIV(+) CCR5(-/-) individuals carrying the CCR5Delta32 59537-A/A variant expressed trace amounts of CCR5Delta32 protein compared to the individuals carrying the CCR5Delta32 59537-G/G genotype. The results imply that the absence of CCR5Delta32 protein in two HIV(+) individuals is due to a genetic defect in the translation of the protein. Together, these results highlight the importance of the CCR5Delta32 protein as an HIV suppressive factor and provide further insight into the mechanism of the protective effect of the CCR5Delta32 mutation.