Selective Neuronal Uptake and Distribution of AAVrh8, AAV9, and AAVrh10 in Sheep After Intra-Striatal Administration.

BACKGROUND: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule. OBJECTIVE: To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep. METHODS: We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain. RESULTS: Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry. CONCLUSIONS: Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.

Effects of HIV-1 TAT protein and methamphetamine exposure on visual discrimination and executive function in mice.

Mild neurocognitive impairments are common in people with human immunodeficiency virus (HIV) infection. HIV-encoded proteins, such as trans-activator of transcription (TAT), contribute to neuropathology and cognitive function in medicated subjects. The combination of TAT and comorbid methamphetamine use may further impair neurocognitive function in HIV-positive individuals by affecting dopaminergic systems in the brain. The current study examined the effects of TAT protein expression and methamphetamine exposure on cognitive function and dopamine systems in mice. Transgenic mice with inducible brain expression of the TAT protein were exposed to a binge methamphetamine regimen. TAT expression was induced via a doxycycline-containing diet during the final stage of the regimen and maintained throughout cognitive testing. Learning and executive function were assessed using an operant visual discrimination protocol, with a strategy switch and reversal. TAT expression and methamphetamine exposure improved visual discrimination learning. Combined TAT expression and methamphetamine exposure increased perseverative errors during reversal learning. TAT expression altered reversal learning by improving early stage, but impairing late stage, learning. TAT expression was also associated with an increase in dopamine transporter expression in the caudate putamen. These results highlight that TAT expression and methamphetamine exposure likely affect a range of selective cognitive processes, with some potentially improving function under certain conditions.

Neuroimaging abnormalities in clade C HIV are independent of Tat genetic diversity.

Controversy remains regarding the neurotoxicity of clade C human immunodeficiency virus (HIV-C). When examined in preclinical studies, a cysteine to serine substitution in the C31 dicysteine motif of the HIV-C Tat protein (C31S) results in less severe brain injury compared to other viral clades. By contrast, patient cohort studies identify significant neuropsychological impairment among HIV-C individuals independent of Tat variability. The present study clarified this discrepancy by examining neuroimaging markers of brain integrity among HIV-C individuals with and without the Tat substitution. Thirty-seven HIV-C individuals with the Tat C31S substitution, 109 HIV-C individuals without the Tat substitution (C31C), and 34 HIV- controls underwent 3T structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). Volumes were determined for the caudate, putamen, thalamus, corpus callosum, total gray matter, and total white matter. DTI metrics included fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD). Tracts of interest included the anterior thalamic radiation (ATR), cingulum bundle (CING), uncinate fasciculus (UNC), and corpus callosum (CC). HIV+ individuals exhibited smaller volumes in subcortical gray matter, total gray matter and total white matter compared to HIV- controls. HIV+ individuals also exhibited DTI abnormalities across multiple tracts compared to HIV- controls. By contrast, neither volumetric nor diffusion indices differed significantly between the Tat C31S and C31C groups. Tat C31S status is not a sufficient biomarker of HIV-related brain integrity in patient populations. Clinical attention directed at brain health is warranted for all HIV+ individuals, independent of Tat C31S or clade C status.

Axonal transport of AAV9 in nonhuman primate brain.

A pilot study in nonhuman primates was conducted, in which two Rhesus macaques received bilateral parenchymal infusions of adeno-associated virus serotype 9 encoding green fluorescent protein (AAV9-GFP) into each putamen. The post-surgical in-life was restricted to 3 weeks in order to minimize immunotoxicity expected to arise from expression of GFP in antigen-presenting cells. Three main findings emerged from this work. First, the volume over which AAV9 expression was distributed (Ve) was substantially greater than the volume of distribution of MRI signal (Vd). This stands in contrast with Ve/Vd ratio of rAAV2, which is lower under similar conditions. Second, post-mortem analysis revealed expression of GFP in thalamic and cortical neurons as well as dopaminergic neurons projecting from substantia nigra pars compacta, indicating retrograde transport of AAV9. However, fibers in the substantia nigra pars reticulata, a region that receives projections from putamen, also stained for GFP, indicating anterograde transport of AAV9 as well. Finally, one hemisphere received a 10-fold lower dose of vector compared with the contralateral hemisphere (1.5 × 10(13) vg ml(-1)) and we observed a much stronger dose effect on anterograde-linked than on retrograde-linked structures. These data suggest that AAV9 can be axonally transported bi-directionally in the primate brain. This has obvious implications to the clinical developing of therapies for neurological disorders like Huntington's or Alzheimer's diseases.

Multilevel analysis of neuropathogenesis of neurocognitive impairment in HIV.

The neuropathogenesis of HIV-associated neurocognitive disorders (HAND) remains puzzling. We interrogated several levels of data (host genetic, histopathology, brain viral load, and neurocognitive) to identify histopathological changes most relevant to HAND. The design of the study is a clinicopathological study employing genetic association analyses. Data and brain tissue from 80 HIV-infected adults were used. Markers in monocyte chemoattractant protein-1 (MCP-1), interleukin 1-alpha (IL1-α), macrophage inflammatory protein 1-alpha (MIP1-α), DRD3, DRD2, and apolipoprotein E (ApoE) were genotyped. Microtubule associated protein 2 (MAP2), synaptophysin (SYP), human leukocyte antigen-DR (HLA-DR), glial fibrillary acidic protein (GFAP), amyloid beta (A-Beta), and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactivity were quantified in the frontal cortex, putamen, and hippocampus. A composite score for each marker (mean of the three brain regions) was used. Neurocognitive functioning and other clinical variables were determined within 1 year of death. Brain HIV RNA viral load was available for a subset of cases. MAP2 and SYP proved most relevant to neurocognitive functioning. Immunoreactivity of these markers, as well as A-Beta and Iba-1, was correlated with brain HIV RNA viral load. Several genetic markers in combination with other factors predicted histopathology: HIV blood viral load, MIP1-α genotype, and DRD3 genotype predicted Iba-1 immunoreactivity; the duration of infection and IL1-α genotype predicted GFAP immunoreactivity; ApoE genotype and age at death predicted A-Beta immunoreactivity. These data indicate that HIV replication in the brain is the primary driving force leading to neuroinflammation and dysfunctional protein clearance, as reflected by A-Beta and Iba-1. Downstream to these changes are synaptodendritic degeneration, which is the immediate histopathological substrate of the neurocognitive impairment characteristic of HAND. These intermediate histopathological phenotypes are influenced by host genetic polymorphisms in genes encoding cytokines/chemokines, neuronal protein clearance pathways, and dopaminergic factors.

Effects of HIV and childhood trauma on brain morphometry and neurocognitive function.

A wide spectrum of neurocognitive deficits characterises HIV infection in adults. HIV infection is additionally associated with morphological brain abnormalities affecting neural substrates that subserve neurocognitive function. Early life stress (ELS) also has a direct influence on brain morphology. However, the combined impact of ELS and HIV on brain structure and neurocognitive function has not been examined in an all-female sample with advanced HIV disease. The present study examined the effects of HIV and childhood trauma on brain morphometry and neurocognitive function. Structural data were acquired using a 3T Magnetom MRI scanner, and a battery of neurocognitive tests was administered to 124 women: HIV-positive with ELS (n = 32), HIV-positive without ELS (n = 30), HIV-negative with ELS (n = 31) and HIV-negative without ELS (n = 31). Results revealed significant group volumetric differences for right anterior cingulate cortex (ACC), bilateral hippocampi, corpus callosum, left and right caudate and left and right putamen. Mean regional volumes were lowest in HIV-positive women with ELS compared to all other groups. Although causality cannot be inferred, findings also suggest that alterations in the left frontal lobe, right ACC, left hippocampus, corpus callosum, left and right amygdala and left caudate may be associated with poorer neurocognitive performance in the domains of processing speed, attention/working memory, abstraction/executive functions, motor skills, learning and language/fluency with these effects more pronounced in women living with both HIV and childhood trauma. This study highlights the potential contributory role of childhood trauma to brain alterations and neurocognitive decline in HIV-infected individuals.

Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

HIV-1 gp120-induced neuroinflammation: relationship to neuron loss and protection by rSV40-delivered antioxidant enzymes.

HIV-1 gp120 neurotoxicity and oxidant injury are well documented, but consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with 100-500 ng HIV-1BaL gp120, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. CD11b-positive microglia were increased 1 day post-challenge; Iba-1- and ED1-positive cells peaked at 7 days and 14 days. Astrocyte infiltration was maximal at 7-14 days. MIP-1alpha was produced immediately, mainly by neurons. ED1- and GFAP-positive cells correlated with neuron loss and gp120 dose. We also tested the effect of more chronic gp120 exposure on neuroinflammation using an experimental model of continuing gp120 exposure. SV(gp120), a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of gp120, ongoing apoptosis in microglia and neurons, and oxidative stress. Increase in microglia and astrocytes was seen following intra-CP SV(gp120) injection, suggesting that continuing gp120 production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced MIP-1alpha and limited neuroinflammation following gp120 administration into the CP, as well as microglia and astrocytes proliferation after injection of SV(gp120) in the striatum. Thus, gp120-induced CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery.

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.

Real-time MR imaging of adeno-associated viral vector delivery to the primate brain.

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

Biostatistical analysis of gene microarrays reveals diverse expression clusters between macaque subspecies in brain SIV infection.

In this study we investigated differences in the gene expression profiling of the brains of rhesus macaques that were uninfected or infected with SIV in the asymptomatic stage or AIDS. The main aim was to use biostatistical methods to classify brain gene expression following SIV infection, without consideration of the biological significance of the individual genes. We also used data from animals treated with different pharmacological substances such as dopaminergic drugs, N-methyl-D-aspartate (NMDA) antagonists or antioxidants during the early stage of infection as these animals exhibited an accelerated or attenuated neuropsychiatric disease progression. We found macaque subspecies to be a more important factor for disease classification based on gene expression profiling than clinical symptoms or neuropathological findings. It is noteworthy that SIV-infected pharmacologically-treated. Chinese animals clustered near uninfected animals independent on the outcome of the treatment, whereas untreated SIV infected animals were clustered in a separate subtree. It is clear from this study that NeuroAIDS is a diverse disease entity and that SIV brain genes can be differentially regulated, depending on the disease type as well as changed dependent on the monkey subspecies.

Human immunodeficiency virus type 1 RNA Levels in different regions of human brain: quantification using real-time reverse transcriptase-polymerase chain reaction.

Human immunodeficiency virus type 1 (HIV-1) enters the central nervous system shortly after the infection and becomes localized in different regions of the brain, leading to various neurological abnormalities including motor disorders and neurocognitive deficits. Although HIV-1-associated functional abnormalities of the central nervous system (CNS) can be evaluated during life by using various test batteries, HIV-1 virus concentration in different brain regions can be measured only after death. The tissues obtained at autopsy provide a valuable source for determining the role of various factors, including that of HIV-1 viral load in the CNS, that may contribute to the regional CNS neuropathogenesis. For this study, we obtained from the National Institutes of Health-sponsored National NeuroAIDS Tissue Consortium (NNTC) the tissues from different brain regions collected at autopsy of HIV-1-positive (N = 38) and HIV-negative (N = 11) individuals, with postmortem intervals of 2 to 29 h, and measured HIV-1 RNA concentration in the frontal cortex, frontal cortex area 4, frontal cortex area 6, basal ganglia, caudate nucleus, putamen, globus pallidus, substantia nigra, and cerebrospinal fluid. Because HIV-1+ individuals were infected with the virus for up to 21 years and the majority of them had used highly active antiretroviral therapy (HAART), we used highly sensitive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay in order to detect a wide dynamic range of HIV-1 RNA with lower detection limit of a single copy. The primers and probes were from the long terminal repeat (LTR) region of HIV genome for achieving higher specificity and sensitivity of detection and amplification. Our results demonstrate a wide variation in the concentration of HIV-1 RNA in different brain regions (5.51 and 8,144,073; log(10) 0.74 and 6.91 copies/g tissue), and despite the high specificity and sensitivity of this method, viral RNA was not detected in 50% of all the samples, and in 30% to 64% of samples of each region of HIV-1+ individuals. However, the highest concentration of viral RNA was found in the caudate nucleus and the lowest concentration in the frontal cortex and cerebrospinal fluid. The viral RNA was undetectable in all samples of HIV-negative individuals.

Up-regulation of glutamate concentration in the putamen and in the prefrontal cortex of asymptomatic SIVmac251-infected macaques without major brain involvement.

We quantified putamen and prefrontal cortex metabolites in macaques with simian immunodeficiency virus infection and searched for virological and histological correlates. Fourteen asymptomatic macaques infected since 8-78 months (median: 38) were compared with eight uninfected ones. Absolute concentrations of acetate, alanine, aspartate, choline, creatine, GABA, glutamate, glutamine, lactate, myo-inositol, N-acetylaspartate, taurine and valine were determined by ex vivo proton magnetic resonance spectroscopy. Glutamate concentration in the CSF was determined by HPLC. Gliosis was assessed by glial fibrillary acidic protein and CD68 immunohistochemistry. Glutamate concentration was slightly increased in the prefrontal cortex (19%, p = 0.0152, t-test) and putamen (13%, p = 0.0354, t-test) of the infected macaques, and was unaffected in the CSF. Myo-inositol concentration was increased in the prefrontal cortex only (27%, p = 0.0136). The concentrations of glutamate and myo-inositol in the prefrontal cortex were higher in the animals with marked or intense microgliosis (p = 0.0114). The other studied metabolites, including N-acetylaspartate, were not altered. Glutamate concentration may thus increase in the cerebral parenchyma in asymptomatic animals, but is not accompanied by a detectable decrease in N-acetylaspartate concentration (neuronal dysfunction). Thus, there are probably compensatory mechanisms that may limit glutamate increase and/or counterbalance its effects.

Brain choline acetyltransferase reduction in SIV infection. An index of early dementia?

HIV infection at late stages is associated with neurological complications including impaired motor and cognitive functions. We used simian immunodeficiency (SIV)-infected rhesus monkeys, an animal model of HIV infection, to investigate changes in choline acetyltransferase (ChAT) activity, a biochemical marker of cognitive function, in post-mortem brains during early, asymptomatic SIV infection and AIDS. ChAT activity was dramatically reduced in putamen and hippocampus already during asymptomatic infection. In animals with AIDS, ChAT activity was further decreased. The reduction of ChAT was not related to brain viral load or CNS pathological lesions. Our results demonstrate deficits in ChAT activity already during the first months of SIV infection and imply that cognitive dysfunction may occur early in immunodeficiency viral infections.

Lentiviral gene transfer to the nonhuman primate brain.

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinson's disease.

Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system.

Mice devoid of PrPC (Prnp%) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnp% brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnp% mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnp% mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

Assessment of neuronal density in the putamen in human immunodeficiency virus (HIV) infection. Application of stereology and spatial analysis of quadrats.

Human immunodeficiency virus causes neuronal loss in various brain regions, but it has not been reported in the putamen. However, decrease in the volume of the putamen has been observed by magnetic resonance imaging. In order to clarify this issue two complementary methods; the stereological probe, the disector, and spatial analysis of quadrats, were applied in nondemented individuals who had died of acquired immune deficiency syndrome. A 21% decrease in neuronal density was observed in the human immunodeficiency virus group, especially those cases with human immunodeficiency virus encephalitis; however the statistical significance of this finding was borderline.