Intercellular viral spread and intracellular transposition of Drosophila gypsy.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.

Prokaryotic SPHINX replication sequences are conserved in mammalian brain and participate in neurodegeneration.

A new class of viral mammalian Slow Progressive Hidden INfections of variable (X) latency ("SPHINX") DNAs, represented by the 1.8 and 2.4 kb nuclease-protected circular elements, were discovered in highly infectious cytoplasmic particles isolated from Creutzfeldt-Jakob Disease (CJD) and scrapie samples. These DNAs contained replication initiation sequences (REPs) with approximately 70% homology to those of environmental Acinetobacter phage. Antibodies against REP peptides from the 1.8 kb DNA highlighted a 41 kDa protein (spx) on Western blots, and in situ studies previously revealed its peripheral tissue expression, for example, in pancreatic islet cells, keratinocytes, kidney tubules, and oocytes but not pancreatic exocrine cells, alveoli, and striated muscle. To determine if spx concentrated in specific neurons and synapses, and also maintained a conserved pattern of architectural organization in mammalian brains, we evaluated mouse, rat, hamster, guinea pig (GP), and human samples. Most outstanding was the cross-species concentration of spx in huge excitatory synapses of mossy fibers and small internal granule neuron synapses, the only excitatory neuron within the cerebellum. Spx also localized to excitatory glutamate type synapses in the hippocampus, and both cerebellar and hippocampal synaptic spx was demonstrable ultrastructurally. Studies of two well-characterized models of sporadic CJD (sCJD) revealed novel spx pathology. Vacuolar loss of cerebellar synaptic complexes, thinning of the internal granule cell layer, and fibrillar spx accumulations within Purkinje neurons were prominent in sCJD GP brains. In rats, comparable spx fibrillar changes appeared in hippocampal pyramidal neurons, and they preceded prion protein misfolding. Hence, spx is an integral player in progressive neurodegeneration. The evolutionary origin, spread, and neuropathology of SPHINX 1.8 REP sequences opens another unanticipated chapter for mammalian symbiotic interactions with environmental microbes.

Porcine Hemagglutinating Encephalomyelitis Virus Activation of the Integrin α5ß1-FAK-Cofilin Pathway Causes Cytoskeletal Rearrangement To Promote Its Invasion of N2a Cells.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dynamics are thought to be closely related to this type of nerve damage. Currently, the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear when PHEV enters the host cells. Here, we demonstrate that entry of PHEV into N2a cells induces a biphasic remodeling of the actin cytoskeleton and a dynamic change in cofilin activity. Viral entry is affected by the disruption of actin kinetics or alteration of cofilin activity. PHEV binds to integrin α5ß1 and then initiates the integrin α5ß1-FAK signaling pathway, leading to virus-induced early cofilin phosphorylation and F-actin polymerization. Additionally, Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42), and downstream regulatory gene p21-activated protein kinases (PAKs) are recruited as downstream mediators of PHEV-induced dynamic changes of the cofilin activity pathway. In conclusion, we demonstrate that PHEV utilizes the integrin α5ß1-FAK-Rac1/Cdc42-PAK-LIMK-cofilin pathway to cause an actin cytoskeletal rearrangement to promote its own invasion, providing theoretical support for the development of PHEV pathogenic mechanisms and new antiviral targets.IMPORTANCE PHEV, a member of the Coronaviridae family, is a typical neurotropic virus that primarily affects the nervous system of piglets to produce typical neurological symptoms. However, the mechanism of nerve damage caused by the virus has not been fully elucidated. Actin is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells. Additionally, the morphological structure and function of nerve cells depend on the dynamic regulation of the actin skeleton. Therefore, exploring the mechanism of neuronal injury induced by PHEV from the perspective of the actin cytoskeleton not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets. This is the first report to define a mechanistic link between alterations in signaling from cytoskeleton pathways and the mechanism of PHEV invading nerve cells.

Zika Virus Replication in Dorsal Root Ganglia Explants from Interferon Receptor1 Knockout Mice Causes Myelin Degeneration.

Zika virus (ZIKV) is a neurotropic agent that targets the developing fetal brain in women infected during pregnancy. In addition to the developing central nervous system, ZIKV has been recently shown to infect cells of the peripheral nervous system (PNS), highlighting its potential to cause acute peripheral neuropathies in adults, such as Guillain-Barré Syndrome (GBS). Here we show that myelinating dorsal root ganglia (DRG) explants obtained from interferon-alpha/beta receptor knock-out mice are productively infected by ZIKV. Virus replication is cytopathic in both peripheral neurons and myelinating Schwann cells leading to myelin disruption. These results confirm and extend previous observations suggesting that the PNS is indeed a potential site of ZIKV infection, replication and cytopathicity.

Persistent EcoHIV infection induces nigral degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-intoxicated mice.

The widespread use of antiretroviral therapy for treatment of human immunodeficiency virus (HIV) infections has dramatically improved the quality and duration of life for HIV-positive individuals. Despite this success, HIV persists for the life of an infected person in tissue reservoirs including the nervous system. Thus, whether HIV exacerbates age-related brain disorders such as Parkinson's disease (PD) is of concern. In support of this idea, HIV infection can be associated with motor and gait abnormalities that parallel late-stage manifestations of PD including dopaminergic neuronal loss. With these findings in hand, we investigated whether viral infection could affect nigrostriatal degeneration or exacerbate chemically induced nigral degeneration. We now demonstrate an additive effect of EcoHIV on dopaminergic neuronal loss and neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. HIV-1-infected humanized mice failed to recapitulate these EcoHIV results suggesting species-specific neural signaling. The results demonstrate a previously undefined EcoHIV-associated neurodegenerative response that may be used to model pathobiological aspects of PD.

Colony Stimulating Factor-1 Receptor Expressing Cells Infiltrating the Cornea Control Corneal Nerve Degeneration in Response to HSV-1 Infection.

Purpose: Herpes simplex virus type-1 (HSV-1) is a leading cause of neurotrophic keratitis, characterized by decreased or absent corneal sensation due to damage to the sensory corneal innervation. We previously reported the elicited immune response to infection contributes to the mechanism of corneal nerve regression/damage during acute HSV-1 infection. Our aim is to further establish the involvement of infiltrated macrophages in the mechanism of nerve loss upon infection. Methods: Macrophage Fas-Induced Apoptosis (MAFIA) transgenic C57BL/6 mice were systemically treated with AP20187 dimerizer or vehicle (VEH), and their corneas, lymph nodes, and blood were assessed for CD45+CD11b+GFP+ cell depletion by flow cytometry (FC). Mice were ocularly infected with HSV-1 or left uninfected. At 2, 4, and/or 6 days post infection (PI), corneas were assessed for sensitivity and harvested for FC, nerve structure by immunohistochemistry, viral content by plaque assay, soluble factor content by suspension array, and activation of signaling pathways by Western blot analysis. C57BL6 mice were used to compare to the MAFIA mouse model. Results: MAFIA mice treated with AP20187 had efficient depletion of CD45+CD11b+GFP+ cells in the tissues analyzed. The reduction of CD45+CD11b+GFP+ cells recruited to the infected corneas of AP20187-treated mice correlated with preservation of corneal nerve structure and function, decreased protein concentration of inflammatory cytokines, and decreased STAT3 activation despite no changes in viral content in the cornea compared to VEH-treated animals. Conclusions: Our results suggest infiltrated macrophages are early effectors in the nerve regression following HSV-1 infection. We propose the neurodegeneration mechanism involves macrophages, local up-regulation of IL-6, and activation of STAT3.

Zika (PRVABC59) Infection Is Associated with T cell Infiltration and Neurodegeneration in CNS of Immunocompetent Neonatal C57Bl/6 Mice.

The recent spread of Zika virus (ZIKV) and its association with increased rates of Guillain Barre and other neurological disorders as well as congenital defects that include microcephaly has created an urgent need to develop animal models to examine the pathogenesis of the disease and explore the efficacy of potential therapeutics and vaccines. Recently developed infection models for ZIKV utilize mice defective in interferon responses. In this study we establish and characterize a new model of peripheral ZIKV infection using immunocompetent neonatal C57BL/6 mice and compare its clinical progression, virus distribution, immune response, and neuropathology with that of C57BL/6-IFNAR KO mice. We show that while ZIKV infected IFNAR KO mice develop bilateral hind limb paralysis and die 5-6 days post-infection (dpi), immunocompetent B6 WT mice develop signs of neurological disease including unsteady gait, kinetic tremors, severe ataxia and seizures by 13 dpi that subside gradually over 2 weeks. Immunohistochemistry show viral antigen predominantly in cerebellum at the peak of the disease in both models. However, whereas IFNAR KO mice showed infiltration by neutrophils and macrophages and higher expression of IL-1, IL-6 and Cox2, B6 WT mice show a cellular infiltration in the CNS composed predominantly of T cells, particularly CD8+ T cells, and increased mRNA expression levels of IFNg, GzmB and Prf1 at peak of disease. Lastly, the CNS of B6 WT mice shows evidence of neurodegeneration predominantly in the cerebellum that are less prominent in mice lacking the IFN response possibly due to the difference in cellular infiltrates and rapid progression of the disease in that model. The development of the B6 WT model of ZIKV infection will provide insight into the immunopathology of the virus and facilitate assessments of possible therapeutics and vaccines.

Loss of cerebellar neurons in the progression of lentiviral disease: effects of CNS-permeant antiretroviral therapy.

BACKGROUND: The majority of investigations on HIV-associated neurocognitive disorders (HAND) neglect the cerebellum in spite of emerging evidence for its role in higher cognitive functions and dysfunctions in common neurodegenerative diseases. METHODS: We systematically investigated the molecular and cellular responses of the cerebellum as contributors to lentiviral infection-induced neurodegeneration, in the simian immunodeficiency virus (SIV)-infected rhesus macaque model for HIV infection and HAND. Four cohorts of animals were studied: non-infected controls, SIV-infected asymptomatic animals, and SIV-infected AIDS-diseased animals with and without brain-permeant antiretroviral treatment. The antiretroviral utilized was 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG), a CNS-permeable nucleoside reverse transcriptase inhibitor. Quantitation of granule cells and Purkinje cells, of an established biomarker of SIV infection (gp41), of microglial/monocyte/macrophage markers (IBA-1, CD68, CD163), and of the astroglial marker (GFAP) were used to reveal cell-specific cerebellar responses to lentiviral infection and antiretroviral therapy (ART). The macromolecular integrity of the blood brain barrier was tested by albumin immunohistochemistry. RESULTS: Productive CNS infection was observed in the symptomatic stage of disease, and correlated with extensive microglial/macrophage and astrocyte activation, and widespread macromolecular blood brain barrier defects. Signs of productive infection, and inflammation, were reversed upon treatment with 6-Cl-ddG, except for a residual low-grade activation of microglial cells and astrocytes. There was an extensive loss of granule cells in the SIV-infected asymptomatic cohort, which was further increased in the symptomatic stage of the disease and persisted after 6-Cl-ddG (administered after the onset of symptoms of AIDS). In the symptomatic stage, Purkinje cell density was reduced. Purkinje cell loss was likewise unaffected by 6-Cl-ddG treatment at this time. CONCLUSIONS: Our findings suggest that neurodegenerative mechanisms are triggered by SIV infection early in the disease process, i. e., preceding large-scale cerebellar productive infection and marked neuroinflammation. These affect primarily granule cells early in disease, with later involvement of Purkinje cells, indicating differential vulnerability of the two neuronal populations. The results presented here indicate a role for the cerebellum in neuro-AIDS. They also support the conclusion that, in order to attenuate the development of motor and cognitive dysfunctions in HIV-positive individuals, CNS-permeant antiretroviral therapy combined with anti-inflammatory and neuroprotective treatment is indicated even before overt signs of CNS inflammation occur.

IL-6 Contributes to Corneal Nerve Degeneration after Herpes Simplex Virus Type I Infection.

Herpes simplex virus type 1 (HSV-1) is a leading cause of neurotrophic keratitis characterized by decreased corneal sensation because of damage to the corneal sensory fibers. We and others have reported regression of corneal nerves during acute HSV-1 infection. To determine whether denervation is caused directly by the virus or indirectly by the elicited immune response, mice were infected with HSV-1 and topically treated with dexamethasone (DEX) or control eye drops. Corneal sensitivity was measured using a Cochet-Bonnet esthesiometer and nerve network structure via immunohistochemistry. Corneas were assessed for viral content by plaque assay, leukocyte influx by flow cytometry, and content of chemokines and inflammatory cytokines by suspension array. DEX significantly preserved corneal nerve structure and sensitivity on infection. DEX reduced myeloid and T-cell populations in the cornea and did not affect viral contents at 4 and 8 days post infection. The elevated protein contents of chemokines and inflammatory cytokines on infection were greatly suppressed by DEX. Subconjunctival delivery of neutralizing antibody against IL-6 to infected mice resulted in partial preservation of corneal nerve structure and sensitivity. Our study supports a role for the immune response, but not local virus replication in the development of HSV-1-induced neurotrophic keratitis. IL-6 is one of the factors produced by the elicited inflammatory response to HSV-1 infection contributing to nerve regression.

Tracking Epidermal Nerve Fiber Changes in Asian Macaques: Tools and Techniques for Quantitative Assessment.

Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus-associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.

Autophagy interaction with herpes simplex virus type-1 infection.

More than 50% of the U.S. population is infected with herpes simplex virus type-I (HSV-1) and global infectious estimates are nearly 90%. HSV-1 is normally seen as a harmless virus but debilitating diseases can arise, including encephalitis and ocular diseases. HSV-1 is unique in that it can undermine host defenses and establish lifelong infection in neurons. Viral reactivation from latency may allow HSV-1 to lay siege to the brain (Herpes encephalitis). Recent advances maintain that HSV-1 proteins act to suppress and/or control the lysosome-dependent degradation pathway of macroautophagy (hereafter autophagy) and consequently, in neurons, may be coupled with the advancement of HSV-1-associated pathogenesis. Furthermore, increasing evidence suggests that HSV-1 infection may constitute a gradual risk factor for neurodegenerative disorders. The relationship between HSV-1 infection and autophagy manipulation combined with neuropathogenesis may be intimately intertwined demanding further investigation.

Central and peripheral markers of neurodegeneration and monocyte activation in HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorders (HAND) affect up to 50 % of HIV-infected adults, independently predict HIV morbidity/mortality, and are associated with neuronal damage and monocyte activation. Cerebrospinal fluid (CSF) neurofilament subunits (NFL, pNFH) are sensitive surrogate markers of neuronal damage in several neurodegenerative diseases. In HIV, CSF NFL is elevated in individuals with and without cognitive impairment, suggesting early/persistent neuronal injury during HIV infection. Although individuals with severe cognitive impairment (HIV-associated dementia (HAD)) express higher CSF NFL levels than cognitively normal HIV-infected individuals, the relationships between severity of cognitive impairment, monocyte activation, neurofilament expression, and systemic infection are unclear. We performed a retrospective cross-sectional study of 48 HIV-infected adults with varying levels of cognitive impairment, not receiving antiretroviral therapy (ART), enrolled in the CNS Anti-Retroviral Therapy Effects Research (CHARTER) study. We quantified NFL, pNFH, and monocyte activation markers (sCD14/sCD163) in paired CSF/plasma samples. By examining subjects off ART, these correlations are not confounded by possible effects of ART on inflammation and neurodegeneration. We found that CSF NFL levels were elevated in individuals with HAD compared to cognitively normal or mildly impaired individuals with CD4+ T-lymphocyte nadirs ≤200. In addition, CSF NFL levels were significantly positively correlated to plasma HIV-1 RNA viral load and negatively correlated to plasma CD4+ T-lymphocyte count, suggesting a link between neuronal injury and systemic HIV infection. Finally, CSF NFL was significantly positively correlated with CSF pNFH, sCD163, and sCD14, demonstrating that monocyte activation within the CNS compartment is directly associated with neuronal injury at all stages of HAND.

Role of neurotrophic factor alterations in the neurodegenerative process in HIV associated neurocognitive disorders.

Migration of HIV infected cells into the CNS is associated with a spectrum of neurological disorders, ranging from milder forms of HIV-associated neurocognitive disorders (HAND) to HIV-associated dementia (HAD). These neuro-psychiatric syndromes are related to the neurodegenerative pathology triggered by the release of HIV proteins and cytokine/chemokines from monocytes/macrophages into the CNS -a condition known as HIV encephalitis (HIVE). As a result of more effective combined anti-retroviral therapy patients with HIV are living longer and thus the frequency of HAND has increased considerably, resulting in an overlap between the neurodegenerative pathology associated with HIV and that related to aging. In fact, HIV infection is believed to hasten the aging process. The mechanisms through which HIV and aging lead to neurodegeneration include: abnormal calcium flux, excitotoxicity, signaling abnormalities, oxidative stress and autophagy defects. Moreover, recent studies have shown that defects in the processing and transport of neurotrophic factors such as fibroblast growth factors (FGFs), neural growth factor (NGF) and brain-derived growth factor (BDNF) might also play a role. Recent evidence implicates alterations in neurotrophins in the pathogenesis of neurodegeneration associated with HAND in the context of aging. Here, we report FGF overexpression curtails gp120-induced neurotoxicity in a double transgenic mouse model. Furthermore, our data show disparities in brain neurotrophic factor levels may be exacerbated in HIV patients over 50 years of age. In this review, we discuss the most recent findings on neurotrophins and HAND in the context of developing new therapies to combat HIV infection in the aging population.

Brain viral burden, neuroinflammation and neurodegeneration in HAART-treated HIV positive injecting drug users.

The long-term impact of chronic human immunodeficiency virus (HIV) infection on brain status in injecting drug users (IDU) treated with highly active antiretroviral therapy (HAART) is unknown. Viral persistence in the brain with ongoing neuroinflammation may predispose to Alzheimer-like neurodegeneration. In this study, we investigated the brains of ten HAART-treated individuals (six IDU and four non-DU), compared with ten HIV negative controls (six IDU and four non-DU). HIV DNA levels in brain tissue were correlated with plasma and lymphoid tissue viral loads, cognitive status, microglial activation and Tau protein and amyloid deposition. Brain HIV proviral DNA levels were low in most cases but higher in HIV encephalitis (n = 2) and correlated significantly with levels in lymphoid tissue (p = 0.0075), but not with those in plasma. HIV positive subjects expressed more Tau protein and amyloid than HIV negative controls (highest in a 58 year old), as did IDU, but brain viral loads showed no relation to Tau and amyloid. Microglial activation linked significantly to HIV positivity (p = 0.001) and opiate abuse accentuated these microglial changes (p = 0.05). This study confirms that HIV DNA persists in brains despite HAART and that opiate abuse adds to the risk of brain damage in HIV positive subjects. Novel findings in this study show that (1) plasma levels are not a good surrogate indicator of brain status, (2) viral burden in brain and lymphoid tissues is related, and (3) while Tau and amyloid deposition is increased in HIV positive IDU, this is not specifically related to increased HIV burden within the brain.

Resveratrol exacerbates both autoimmune and viral models of multiple sclerosis.

The polyphenol compound resveratrol is reported to have multiple functions, including neuroprotection, and no major adverse effects have been reported. Although the neuroprotective effects have been associated with sirtuin 1 activation by resveratrol, the mechanisms by which resveratrol exerts such functions are a matter of controversy. We examined whether resveratrol can be neuroprotective in two models of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). EAE was induced in C57BL/6 mice, which were fed a control diet or a diet containing resveratrol during either the induction or effector phase or through the whole course of EAE. SJL/J mice were infected with TMEV and fed a control diet or a diet containing resveratrol during the chronic phase of TMEV-IDD. In EAE, all groups of mice treated with resveratrol had more severe clinical signs than the control group. In particular, resveratrol treatment during the induction phase resulted in the most severe EAE, both clinically and histologically. Similarly, in the viral model, the mice treated with resveratrol developed significantly more severe TMEV-IDD than the control group. Thus, surprisingly, the resveratrol treatment significantly exacerbated demyelination and inflammation without neuroprotection in the central nervous system in both models. Our findings indicate that caution should be exercised in potential therapeutic applications of resveratrol in human inflammatory demyelinating diseases, including multiple sclerosis.

Borna disease virus-induced neuronal degeneration dependent on host genetic background and prevented by soluble factors.

Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.

Viral models of multiple sclerosis: neurodegeneration and demyelination in mice infected with Theiler's virus.

Multiple sclerosis (MS) is a complex inflammatory disease of unknown etiology that affects the central nervous system (CNS) white matter, and for which no effective cure exists. Indeed, whether the primary event in MS pathology affects myelin or axons of the CNS remains unclear. Animal models are necessary to identify the immunopathological mechanisms involved in MS and to develop novel therapeutic and reparative approaches. Specifically, viral models of chronic demyelination and axonal damage have been used to study the contribution of viruses in human MS, and they have led to important breakthroughs in our understanding of MS pathology. The Theiler's murine encephalomyelitis virus (TMEV) model is one of the most commonly used MS models, although other viral models are also used, including neurotropic strains of mouse hepatitis virus (MHV) that induce chronic inflammatory demyelination with similar histological features to those observed in MS. This review will discuss the immunopathological mechanisms involved in TMEV-induced demyelinating disease (TMEV-IDD). The TMEV model reproduces a chronic progressive disease due to the persistence of the virus for the entire lifespan in susceptible mice. The evolution and significance of the axonal damage and neuroinflammation, the importance of epitope spread from viral to myelin epitopes, the presence of abortive remyelination and the existence of a brain pathology in addition to the classical spinal cord demyelination, are some of the findings that will be discussed in the context of this TMEV-IDD model. Despite their limitations, viral models remain an important tool to study the etiology of MS, and to understand the clinical and pathological variability associated with this disease.

Infectious agents and neurodegeneration.

A growing body of epidemiologic and experimental data point to chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Infections of the central nervous system, especially those characterized by a chronic progressive course, may produce multiple damage in infected and neighbouring cells. The activation of inflammatory processes and host immune responses cause chronic damage resulting in alterations of neuronal function and viability, but different pathogens can also directly trigger neurotoxic pathways. Indeed, viral and microbial agents have been reported to produce molecular hallmarks of neurodegeneration, such as the production and deposit of misfolded protein aggregates, oxidative stress, deficient autophagic processes, synaptopathies and neuronal death. These effects may act in synergy with other recognized risk factors, such as aging, concomitant metabolic diseases and the host's specific genetic signature. This review will focus on the contribution given to neurodegeneration by herpes simplex type-1, human immunodeficiency and influenza viruses, and by Chlamydia pneumoniae.

HIV-1 gp120 enhances outward potassium current via CXCR4 and cAMP-dependent protein kinase A signaling in cultured rat microglia.

Microglia are critical cells in mediating the pathophysiology of neurodegenerative disorders such as HIV-associated neurocognitive disorders. We hypothesize that HIV-1 glycoprotein 120 (gp120) activates microglia by enhancing outward K(+) currents, resulting in microglia secretion of neurotoxins, consequent neuronal dysfunction, and death. To test this hypothesis, we studied the effects of gp120 on outward K(+) current in cultured rat microglia. Application of gp120 enhanced outward K(+) current in a dose-dependent manner, which was blocked by voltage-gated K(+) (K(v) ) channel blockers. Western blot analysis revealed that gp120 produced an elevated expression of K(v) channel proteins. Examination of activation and inactivation of outward K(+) currents showed that gp120 shifted membrane potentials for activation and steady-state inactivation. The gp120-associated enhancement of outward K(+) current was blocked by either a CXCR4 receptor antagonist T140 or a specific protein kinase A (PKA) inhibitor H89, suggesting the involvement of chemokine receptor CXCR4 and PKA in gp120-mediated enhancement of outward K(+) current. Biological significance of gp120-induced enhancement of microglia outward K(+) current was demonstrated by experimental results showing the neurotoxic activity of gp120-stimulated microglia, evaluated by TUNEL staining and MTT assay, significantly attenuated by K(v) channel blockers. Taken together, these results suggest that gp120 induces microglia neurotoxic activity by enhancing microglia outward K(+) current and that microglia K(v) channels may function as a potential target for the development of therapeutic strategies.

Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration.

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.