Brandon K. Harvey - US grants

Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. We characterized seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1, 5, 6, 7 and 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using LDH and MTS assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity. We are now using these infection parameters to deliver potentital therapeutic genes in vitro models of methamphetamine-toxicity, excitotoxicity, hypoxia and Parkinsons disease as well as characterize the signal transduction pathways involved in the protective and regenerative properties of bone morphogenetic proteins (BMPs). Our second approach uses AAV vectors for in vivo delivery to the brain in models of stroke, Parkinsons disease and methamphetamine-toxicity. Specifically, we evaluate the protective and regenerative properties of BMP7 and its signaling molecules. Currently, we are testing the role of AAVBMP7 and AAVSMADs in models of Parkinsons and methamphetamine-toxicity.

We generated an AAV vector expressing the human mu opioid receptor (huMOR) and are evaluating the role of huMOR in methamphetamine sensitization in mice. Our preliminary findings show that huMOR expression by an AAV vector in specific brain regions alters methamphetamine sensitization. We have submitted this work for publication and it is currently under review. We have also generated an AAV vector expressing the glutamate transporter (GLT-1) and demonstrated that it functional. We have ongoing work examining the ability of GLT-1 to reduce damage caused by ischemia using a rat model of stroke. The protection against ischemia was accompanied by a decrease in ischemia-induced glutamate overflow as measured by microdialysis and this study was published this year. AAV-GLT-1 was created to modulate the levels of extracellular glutamate. We have begun experiments examining the ability of excess GLT-1 overexpression by AAV to reduce excitoxicity by glutamate. We have also begun examining the GLT-1 overexpression in specfic brain regions for alterations to methamphetamine sensitization. These experiments are ongoing. Lastly, we collaborated with Dr. Bruce Hope (NIDA IRP) to the development of assay to isolate neurons from rodent brain using FACS to understand the cell-specific induced changes following cocaine exposure.

Over the past several years, our group has been focused on studying genes with neuromodulatory, neuroprotective and/or neuroregenerative effects in models of neurodegeneration and neurotoxicity. We have continue to focus on a protein with neuroprotectie properties, mesencephalic astrocyted derived neurotrophic factor (MANF). We published a study last year describing the neuroprotective actions of MANF against ischemic brain injury. We are continuing to explore the neuroprotective and neuroregenerative mechanism(s) MANF. Towards this goal, we published a paper this year describing how the C-terminal tail of the MANF protein is sufficient to confer ER localization and release of secreted protein in response to calcium depletion. In collaboration with Dr. Mart Saarma, we coauthored a paper describing the MANF knockout mouse and the role of MANF in beta cell survival in the pancreas. These data further expand our understadning of the broad protective functions of MANF. We are also preparing a manuscript describing the function of MANF in C. elegans as it relates to cellular stress, specifically, endoplasmic reticulum stress. The phenomenon of ER stress occurs in many diseases beyond neurodegenerative and understanding its role may lead to broader therapeutic strategies for MANF and CDNF. Based on findings in our recently published MANF study, we have now published a manuscript describing how the C-terminal ASARTDL sequence of MANF can regulate protein secretion in an ER calcium-dependent manner and have filed a patent for its use as a diagnostic and therapeutic tool. Part of our current research is understanding more about the mechanisms involved in this peptide motif and trying to create proteins that are released in response to ER calcium depletion. We have collaborated with NCATS to develop a screen for drugs that affect ER calcium homeostasis. Thus far, they have screened 15,000 compounds and we have identified 30 compounds that we are following up. Our findings have great potential for developing tools to further our understanding of the relationship between ER calcium and pathogenesis, using ER Ca2+ levels to monitor disease states, and possibly regulating the release of therapeutic proteins. Our group has previously demonstrated the ability of Bone Morphogenetic Protein 7 (BMP7) to promote neuroregeneration. As a continuation of this work, we have used an in vitro model of primary cortical neurons to show that BMP7 changes the appearance of axons and dendrites and have linked this to the change in extracellular matrix proteins. We are preparing the manuscript for publication. In collaboration with Dr. Yun Wang (formerly of NIDA-IRP), we evaluated the neuroregenerative properties of BDNF in rat model of stroke and the results were published this year.

We have the methods and facilities to package and purify both adeno-associated viral (AAV) vectors and lentiviral vectors (LV) for gene delivery studies. We have been making AAV vectors for over 7 years and have begun working with lentiviral vectors. We are routinely generate new vectors both for our lab and for collaborations. The vectors are designed primarily for studies focused on studying the the molecular mechanisms of addiction and neurodegeneration. For example, we have well characterized and novel neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic protein 7 (BMP7), mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopaminerigic neurotrophic factor (CDNF). We also work with neuromodulatory genes such as neurotransmitter tranporters and receptors. Our vectors are available for intramural and extramural collaborators.

The nematode, Caenorhabditis elegans has become an invaluable model organism for studying molecular and cellular functions. We are employing C. elegans to study human gene function as it relates to addiction. We are interested in how allelic variations in genes involved in the biosynthesis, transport, release, reuptake, signaling and catabolism of the neurotransmitter dopamine play a role in the susceptibilities to drugs of abuse such as methamphetamine. In humans and other mammals, dopamine is the primary neurotransmitter of the "reward pathway" which reinforces behaviors that are beneficial to the survival of an organism. It is the reward pathway that is most affected by drugs of abuse as they reinforce their consumption and associated behaviors. C. elegans has eight dopamine neurons that control specific movement and reproductive behaviors. This past year, we have adopted a swimming induced paralysis assay (SWIP) assay and developed a movement tracking assay to examine behavioral response to methamphetamine. We have also used this first year to acquire equipment and strains of C. elegans for our studies. We have hired a research associate with 7 years of experience working with C. elegans and associated molecular biology and transgenic techniques. During our summer students fellowship, she was able to acquire data on dose-dependent methamphetamine-induced changes in behavior. We are in the process of completing the study for publication. The presence of conserved genes involved in dopamine biology coupled with defined behaviors associated with these 8 dopamine neurons allows for testing human gene function in response to drugs of abuse. The ease of genetic manipulation in C. elegans allows us to replace the C. elegans gene such as the dopamine transporter with variants of human dopamine transporter. The "humanized" worms can then be analyzed for changes in drug response. Ultimately, by making genetically tagged versions of the human genes, we can screen for altered behavior after drug exposure and identify the combination of human alleles. We hypothesize that specific combinations of human gene variants or alleles can mediate altered responses to drugs of abuse and will indicate "vulnerabilities" to drugs such as methamphetamine or cocaine. From these studies, we hope use genetic based vulnerabilities to develop more individualized approaches to treating addiction. Towards these goals, we have acquired several variants of the human dopamine transporter with known differences in affinity and transport. We are in the process of generating transgenic worms on a worm dopamine transporter knockout background. In addition to studying gene function and drugs of abuse in the dopaminergic system, we can use models of dopaminergic neurodegeneration to study potential molecules important for dopaminergic neuron development, survival and regeneration of fibers. For example, we have been working on a conserved gene, mesencephalic astrocyte-derived neurotrophic factor (MANF) which was recently identified as a dopaminergic neurotrophic factor in mammals. From our studies in C elegans, we have been able to identify possible mechanisms of neuroprotection for this protein. This past year, we have 1) generated several transgenic animals that demonstrate the cellular location of MANF, 2) characterized MANF knockout worms, 3) generated and characterized antibodies for studying MANF in worms, and 4) identified a novel response to drugs that induce cellular stress. Some of our work was presented at the NIDA MiniSymposium at the 2009 Society for Neurosciene Meeting in Chicago. We are currently preparing the manuscript summarizing our findings of MANF. From our work with C.elegans and MANF, we will return to our mammalian models of Parkinsons disease and methamphetamine toxicity to examine new therapeutic approaches to treating the degeneration of dopaminergic neurons. In summary, we are able to employ C. elegans as a model for human gene function in dopaminergic neurons which are affected in addiction and Parkinsons disease.

We now have staff who are specilized in the methods to package, purify and characterize (in vitro and in vivo) both adeno-associated viral (AAV), lentiviral vectors (LV)and Canine adenoviral vectors (CAV2). We routinely generate vectors both for the NIDA IRP as well as other intramural and extramural collaborations. The vectors are designed primarily for studies focused on molecular mechanisms of addiction and neurodegeneration. For example, we have produced over 20 different vectors for delivery of the opsins for optogenetic manipulation of neuronal circuits. We have also been developing several novel vectors to express synaptically targeted fluorescent proteins, calcium-sensitive fluorescent proteins, infrared reporter protein and photosensitizing proteins for lesioning neurons with light. We have successfully created but not yet published 12 transgenic rat lines including a conditional-mCherry fluorescent reporter rat that will only express the mCherry fluorescent protein where Cre recombinase is present. This rat has been crossed with our DAT-Cre rat to verify expression of Cre in dopaminergic neurons. We also have a transgenic rat line expressing Tet repressor protein. The Tet repressor protein can bind to specific DNA elements (Tet operator) to prevent gene expression. By giving animals tetracycline or an analog we can cause the repressor to come off the DNA and allow gene expression. This will be used in combination with viruses or other rats that containing the Tet operator to allow us to activate gene expression. We have additional Cre-driver rats in early stages of characterization as well as other reporter rats. All of our characterized and published vectors are available for intramural and extramural collaborators.

The recently formed Glia-Neuron Interaction Lab is examining how rodent microglia respond to methamphetamine in both a chronic and acute exposure model. Ongoing work has identified dosing of methamphetamine that cuases unique activation of microglia in the striatum that can be used to examine how activated microglia can affect the surround neurons. A second ongoing project that has been conducted in collaboration with Dr. Jonathan Karn at Case Western University has show that methamphetamine can act directly on a human microglial cell line to activate NFkappaB signaling and increase the transcriptional activity of the HIV LTR promoter. Ongoing studies will examine the signaling by which methamphetamine can influence the NFkB signaling and HIV transcription in microglia.

DESCRIPTION (provided by applicant): One in three HIV-infected individuals develops some form of HIV-associated neurocognitive disorder (HAND). Consumption of drugs of abuse such as methamphetamine (METH) aggravates the symptoms of HAND, but the cellular and molecular mechanisms by which these drugs impact HIV disease progression in the central nervous system (CNS) remain ill-defined. In this study will test the hypothesis that HAND arises from the intermittent reactivation of latently infected microglial cells leading to the expression f the neurotoxic viral proteins, Tat and gp120. Specifically, we will identify the specific contributon of specific molecular networks of glial cells that are involved in the control of HIV latency and te acquisition of an anti-inflammatory M2 phenotype. Our recent unbiased human shRNA library screen for factors that are required to maintain HIV latency in CHME-5/HIV cells showed that HIV silencing in microglila cells can be induced by the ligand- activated nuclear receptors (LA-NR) PPAR, RAR and RXR. Chemical screens of PPAR, RAR and RXR agonists and antagonists confirmed that these receptors have a critical regulatory role in controlling HIV latency in microglial cells and that receptor agonists are potent blockers of HIV reactivation. In this study we will study the molecular basis for LA-NR control of HIV transcription in microglial cells and how this regulatory pathway is modified by METH. Using novel co-culture systems between latently infected microglial cells and neurons we will study the impact METH on the induction of latent HIV proviruses and their subsequent neurotoxicity. We will also use this system to evaluate the neuroprotective effects of PPAR, RAR and RXR agonists, consistent with the recent demonstration that the RXR agonist bexarotene or the PPAR agonist pioglitazone are neuroprotective in mouse models of Alzheimer's disease. Finally we will evaluate the role of the ligand nuclear receptor pathway on HIV induced neurodegeneration using a novel double transgenic mouse model, JR-CSF/hu-CycT1 mice, that permits HIV replication in mouse cells and allows study of how HIV infection induces brain dysfunction. We also plan to develop a novel model for HIV-induced neurodegeneration using latently infected mouse monocytes to repopulate the brains of CD11b-HSVTK transgenic mice where microglial cells have been depleted. The multidisciplinary experiments described in this application represent a comprehensive evaluation of the molecular basis for HIV latency in microglial cells in the brain, the impact of METH on HIV latency and neurodegeneration, and extensive evaluations of the potential of nuclear receptor agonists as neuroprotective agents. We believe that the systematic studies we have proposed here will set the stage for improved clinical management of HIV-associated dementia (HAD) and minor cognitive motor disorder (MCMD) in our patients who abuse drugs.

The Glia-Neuron Interaction Lab is examining how rodent microglia respond to methamphetamine in both a chronic and acute exposure model. Ongoing work has identified dosing of methamphetamine that causes unique activation of microglia in the striatum that can be used to examine how activated microglia can affect the surround neurons. A second ongoing project that has been conducted in collaboration with Dr. Jonathan Karn at Case Western University has show that methamphetamine can act directly on a human microglial cell line to activate NFkappaB signaling and increase the transcriptional activity of the HIV LTR promoter. Ongoing studies will examine the signaling by which methamphetamine can influence the NFkB signaling and HIV transcription in microglia. As part of this collaboration and in collaboration with Drs. Stephen Dewhurst and Harris Gelbard at University of Rochester, we are developing a transgenic rat model of HIV pathogenesis in microglia and astrocytes.

Using a the SERCaMP technology that we previously developed, we found that both a cafeteria diet and high fat diet cause ER calcium dysfunction in rat liver. The dysregulation of ER calcium is implicated in wide variety of disease states. Our findings provide a possible mechanism by which liver function is altered in response to excessive fat in the diet. Our findings our published in the Journal of Hepatology. Based on our SERCaMP technology, we developed a library to screen the human proteome for other C-terminal tails that may confer ER retention and release in response to ER calcium depletion. We identified the tail of an endogenous esterase that functions as a SERCaMP tail and we developed an assay to measure the esterase in the cell culture media and plasma from both humans and rodents. We published a paper describing this novel biomarker of ER calcium depletion in Biomarkers. We describe the characterization and development of multiple transgenic tools that express a near-infrared fluorescent protein (IRFP) in neurons as a reporter of transgene expression. The longer wavelength of light used for visualization offers many advantages over other fluorescent reporters used for neuroscience applications related to our work on the cellular mechanisms of neuronal dysfunction. This work was published in J Neuroscience Methods. In collaboration with Dr. Mikko Airavaara, we published two papers on potential post-stroke therapies. First, we showed that MANF promotes recovery from stroke when administered post-stroke (Sci Adv 2018). We also show that intranasal delivery of naloxone enantiomer was able to reduce stroke-related inflammation in the brain and promote recovery in rat model of stroke (eNeuro 2018). In collaboration with Kent Hamra, we developed a spermatogonial stem cell line from Long Evans rats to be used for generating transgenic rat lines.