Richard Jay Smeyne, Ph.D. - US grants

disease /disorder model; laboratory mouse; tissue /cell culture

DESCRIPTION (provided by applicant): PD is a debilitating neurological disorder that strikes 20 per 100,000 persons greater than 50 years of age. The cause of >90% of all PD cases are unknown. However, the discovery of the meperidine by-product 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has provided a useful model of Parkinsonism that appears to recapitulate the pathology of the disease seen in man. Exposure to this prototypical "environmental toxin" causes a selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). MPTP is a lipophillic molecule that rapidly enters the brain and is metabolized to MPP+ through a series of intermediates to MPP+ by the enzyme MAO-B. MPP + is a substrate for dopamine uptake mechanisms and it accumulates intraneuronally and interferes with complex I of the electron transport chain. We have recently shown that the glial cell is the critical cell for conferring protection or susceptibility to this toxin. Since PD is progressive, both in terms of cell loss and symptomotology, it would be of tremendous clinical value if there were cell biological, pharmacological or non-pharmacological methods that could attenuate cell loss; with or without interruption of the disease triggers. Alternatively, at the least, it would be important to slow the progression of cell loss once symptoms arose. There is a significant literature, dating back to the late 1700's that altering an animals' environment can lead to neurological changes. These changes are manifested as increased brain size, increased learning, and recently it has been shown that environment can increase neurogenesis. Recently, we have preliminary data to suggest that mice raised in an "Enriched Environment" (EE) are protected from MPTP toxicity. In this application, we will study and further establish the EE model. In addition, we will examine if the components (exercise, alterations in environmental complexity and/or social interactions) of the EE can confer neuroprotection. In addition, we will examine the role of the neurotrophin BDNF in EE-dependent neuroprotection. The work proposed and subsequent results generated in the application will be used as pilot data. We believe that the EE model may provide a new approach to prevention of PD symptomatology as well as other neurodegenerative disorders.

DESCRIPTION (provided by applicant): The etiology of Parkinson's disease is multivariate, ranging from identified genetic mutations to strict environmental causation. Parkinson's disease can also occur following exposure to viruses, including the influenza virus. Most influenza infection in humans result in upper respiratory tract infection, but occasionally the brain is affected. At this time, there is considerable threat of a worldwide pandemic from the H5N1 strain of influenza virus, the so called "bird-flu". Previously, the great influenza pandemic of 1918, caused by an H1N1 influenza virus, affected 25-30% of the world's population, killing more than 40 million people. In the surviving population, the pandemic was linked to an outbreak of encephalitis lethargica (Von Economo's disease) followed by a spike in post-encephalic Parkinson's disease. Our preliminary results show that the H5N1 virus is neurotropic and can infect a variety of CNS and PNS areas. However, it is not known if the H5N1 virus can induce specific neurological damage. Since the H5N1 virus has the potential to mutate and start human-to-human transmission (likely leading to worldwide pandemic flu), it is critical to examine-in a mammalian species-if this virus has the ability to contribute to postencephalic neurodegenerative disease with particular emphasis on induced parkinsonism. In this application we propose three specific aims. Specific Aim 1 will determine the time-course for H5N1 influenza virus infection in the central, peripheral and enteric nervous systems of mice after intranasal inoculation. Here we will use immunohistochemical detection of H5N1 virus to map the presence of H5N1 at 1, 3, 7, 10, 21, 28, 60 and 90 days following intranasal administration of H5N1. We will also examine what cells get infected by H5N1 (neurons, astrocytes, microglia, epithelial cells) as well as determine how the virus enters the nervous system. Specific aim 2 will empirically determine if H5N1 influenza virus infection results in a loss of SNpc neurons or depletion of dopamine in its target the striatum as well as if the presence of H5N1 induces an immunological reaction, and Specific Aim 3 will determine if prior CNS infection with H5N1 influenza virus increases the sensitivity to the parkinsonian toxin MPTP. PUBLIC HEALTH RELEVANCE: There are great uncertainties about the timing, virulence, and general scope of a future human influenza pandemic. However, H5N1 has recently demonstrated considerable pandemic potential. There are 3 characteristics that a pandemic influenza strain must have: 1) it must be a new strain of influenza virus never seen in human populations before 2) it must be able to replicate and cause diseases in humans 3) it must transmit efficiently from human to human. H5N1 already meets the first 2 characteristics. Additionally, it is now spreading worldwide through avian populations, possibly becoming endemic in many regions and thereby increasing the risk of human exposure. If an H5N1 human pandemic were to occur, the impact on human populations would be enormous. Estimates of projected casualties vary tremendously, from 2 to 360 million worldwide, depending on assumptions about the lethality of the virus which can vary greatly as the virus adapts to humans (WHO website). However, what is clear is that a significant proportion of the world's population would become infected. Based upon reports of H5N1 neurotropism, including our preliminary studies (see below), an Op-Ed in the New York Times by of Oliver Sacks become particularly prophetic 38. Here, he writes " ...we would do well to reawaken ourselves to what may be a formidable threat not only to human lives, but also to the human brain in those who will survive an H5N1 influenza infection".

DESCRIPTION (provided by applicant): The etiology of Parkinson's disease is multivariate, ranging from identified genetic mutations to strict environmental causation. One environmental agent that has been shown to induce Parkinsonism is virus, including influenza. Our lab, and others, has shown that the highly pathogenic avian influenza virus, H5N1, is neurotropic and can induce a number of Parkinsonism pathologies including loss of the DA phenotype in DA neurons, increased activation of the immune system and increased phosphorylation and aggregation of alpha- synuclein. What is unknown, at this time, is whether the induced Parkinsonian pathologies that we observed are specific to only certain strains of influenza (those known to be neurotropic (H5N1)), or can also be induced by those that do not enter the CNS, including the 1918-H1N1 influenza virus that was responsible for the Spanish flu and has been implicated in von Economo's encephalopathy and the H1N1 influenza virus strain (A/H1N1/CA/04/2009) most responsible for the 2009 pandemic. We also seek to determine the critical gene(s) responsible for influenza neurotropism. In this application, we propose 3 specific aims to investigate the role of influenza in neurodegenerative disease, concentrating on pathologies observed in Parkinson's disease. These are: 1) Identify strains of Type A Influenza that are/are not neurotropic, and identify the gene(s) within the influenza genome that confer neurotropism, 2) Determine the immune response in brain initiated by neurotropic and non-neurotropic influenza viruses, and 3) Determine if the parkinsonian pathologies induced by the H5N1 Type A Influenza virus are common to other Type A influenza viruses. The results of these studies, which utilize a unique series of chimeric influenza viruses generated between the neurotropic H5N1 and 1918-H1N1and 2009 H1N1 influenza viruses, will allow us to identify the key components within the virus that allow entry into the nervous system and understand how peripheral infection can signal activation of the immune response in brain. In terms of human health, these studies will allow us to predict how current (i.e. H5N1 and H1N1) and future emerging influenza strains will impact the CNS; as well as identify targets (both known and unknown) that allow for development of therapeutic agents to interfere with these processes. PUBLIC HEALTH RELEVANCE: More than 25-50 million cases of the flu are reported each year. A number of these influenza viruses are capable of infecting the brain, resulting in a number of neurological symptoms, including altered consciousness, disorientation, seizure, and as well as neurodegenerative disorders such as encephalitic parkinsonism. In this application we propose studies to understand how influenza can enter the nervous system as well as initiate an inflammatory response. In terms of human health, these studies will allow us to predict how current (i.e. H5N1 and H1N1), and future emerging influenza strains will impact the CNS; as well as identify targets (both known and unknown) that allow for development of therapeutic agents to interfere with these processes.

DESCRIPTION (provided by applicant): The etiology of Parkinson's disease is multivariate, ranging from identified genetic mutations to strict environmental causation. One environmental agent that has been shown to induce Parkinsonism is virus, including influenza. Our lab, and others, has shown that the highly pathogenic avian influenza virus, H5N1, is neurotropic and can induce a number of Parkinsonism pathologies including loss of the DA phenotype in DA neurons, increased activation of the immune system and increased phosphorylation and aggregation of alpha- synuclein. What is unknown, at this time, is whether the induced Parkinsonian pathologies that we observed are specific to only certain strains of influenza (those known to be neurotropic (H5N1)), or can also be induced by those that do not enter the CNS, including the 1918-H1N1 influenza virus that was responsible for the Spanish flu and has been implicated in von Economo's encephalopathy and the H1N1 influenza virus strain (A/H1N1/CA/04/2009) most responsible for the 2009 pandemic. We also seek to determine the critical gene(s) responsible for influenza neurotropism. In this application, we propose 3 specific aims to investigate the role of influenza in neurodegenerative disease, concentrating on pathologies observed in Parkinson's disease. These are: 1) Identify strains of Type A Influenza that are/are not neurotropic, and identify the gene(s) within the influenza genome that confer neurotropism, 2) Determine the immune response in brain initiated by neurotropic and non-neurotropic influenza viruses, and 3) Determine if the parkinsonian pathologies induced by the H5N1 Type A Influenza virus are common to other Type A influenza viruses. The results of these studies, which utilize a unique series of chimeric influenza viruses generated between the neurotropic H5N1 and 1918-H1N1and 2009 H1N1 influenza viruses, will allow us to identify the key components within the virus that allow entry into the nervous system and understand how peripheral infection can signal activation of the immune response in brain. In terms of human health, these studies will allow us to predict how current (i.e. H5N1 and H1N1) and future emerging influenza strains will impact the CNS; as well as identify targets (both known and unknown) that allow for development of therapeutic agents to interfere with these processes.

Abstract A number of genes, when mutated, have been shown to underlie the development of Parkinson?s disease (PD), including the Leucine Rich Repeat Kinase 2 gene (LRRK2). A number of studies have identified several mutations in the LRRK2 gene; each which result in an autosomal-dominant late-onset form of PD that have clinical and pathological phenotypes similar to idiopathic PD. Population studies have demonstrated that mutations in the LRRK2 gene account for approximately 13% of all diagnosed familial cases of PD and 5-10% of apparently sporadic PD cases. The two most common LRRK2 mutation, G2019S and R1441G, are located in the MAPKK and ROC GTPase domains, respectively. However, despite intensive research efforts, the precise mechanisms underlying LRRK2-induced parkinsonian pathology are still unclear. Additionally, low penetrance of LRRK2 mutations in humans, and the absence of an obvious neurological phenotype in LRRK2 animal models suggest that this form of PD may result from a complex interplay of genetic predispositions and persistent exogenous insults. preliminary studies in this application support a finding that R1441G and G2019S mutations in LRRK2 affect the functional crosstalk between the peripheral immune system and microglia of the innate immune system in the CNS. This alteration appears to mediate inflammation in the substantia nigra pars compacta (SNpc) and degeneration of SNpc DA neurons. based on our studies, and others in the literature, we hypothesize that neuroinflammation in the CNS of LRRK2 mice following an exogenous insult is not initiated by a direct effect on the brain?s intrinsic immune system (microglia) or through the actions of T-cells that have extravastated from the circulatory system into brain, but are triggered by dysregulated expression of peripheral circulating inflammatory molecules, including cytokines that can pass from the periphery into the brain. In this high risk/high reward application, we propose two specific aims to test this hypothesis. In SA1, we will use bone marrow transplantation to generate mice that express wt LRRK2 expression in T- and B-cells while all other cells (including neurons and microglia) express mutant LRRK2. In SA2, we will use these mice to determine if the replacement of mutant LRRK2 with wt LRRK2 in peripheral T- and B-cells diminishes the response to LPS-induced neuroinflammation and rescues the SNpc DA neuron loss seen in LPS-exposed mutant LRRK2 mice. If mice carrying wt T- and B-cells do not show pathological effects of LPS in the CNS, this will support the hypothesis that a major monogenic cause of PD is immune mediated rather than a direct effect of LRRK2 in the CNS; supporting a paradigm shift in our understanding of the etiology of PD as well as provide new targets for the treatment (and possibly prevention) of PD.

Abstract The etiology of Parkinson?s disease is multivariate, ranging from identified genetic mutations to strict environmental causation. So far, more than 18 genes have been identified that result in parkinsonism. The two most common genetic mutations that lead to parkinsonism are: 1) mutations in the GBA gene that encodes the glucocerebrosidase protein, and 2) mutations in the LRRK2 gene that Leucine Rich Repeat Kinase II protein. In addition to their known ?genetic i.e familial? relationship to disease causation, both the GBA and LRRK2 genes are also considered to be ?risk factors? for development of PD, in that not everyone with these mutations develops Parkinson?s disease and they may only manifest after a second ?hit?. No matter the initiating cause of PD, almost all cases of Parkinson?s disease share common aspects of pathology, including: 1) the presence of aggregated alpha-synuclein, 2) loss of SNpc DA neurons and 3) an increase in neuroinflammation. Additionally, one also sees cognitive and motor output changes. In this application, the we will examine different pathological mechanisms known to initiate Parkinson?s disease, including protein kinase activation, protein management or inflammation will alter/affect the aggregation and spread of ?-syn throughout the nervous system. Specifically, we will examine the effect on PD pathophysiology including SNpc DA neuron loss, loss of basal ganglia catecholamines, induction of neuroinflammation and spread of misfolded alpha-synuclein. We will also examine if cognitive and motor behavioral changes occur in these 3 conditions after PFF seeding. These parkinsonian pathologies will be examined following injection of preformed filaments of alpha-synuclein (PFFs) into three different regions of the CNS, including two known to be involved in PD (olfactory bulb and striatum) and one that is not (internal control, hippocampus). In Specific Aim 1, we will examine if PFFs injected into different regions of the CNS of mice carrying a G2019S mutation in the LRRK2 gene alter the seeding and spread of ?-Syn as well as alter other known pathologies in PD as described above. In Specific Aim 2, we will examine if preformed fibrils of alpha-synuclein (PFFs) injected into different regions of the CNS of mice carrying a L444P GBA mutation alters the seeding and spread of ?-Syn as well as alter other known pathologies in PD as described above. In Specific Aim 3 we will test the hypothesis that a prior neuroinflammatory insult (infection with the H1N1 influenza virus) to the brain will increase the seeding and spread of PFFs in mice carrying PD susceptibility genes as well as alter other known pathologies in PD as described above. These three aims will allow us to determine if any one or more of these pathological mechanisms (kinase activation (genetic), protein mishandling (gene x environment? or viral infection (environment) directly influence the spread of misfolded alpha-synuclein and other common parkinsonian pathologies.

ABSTRACT Approximately16.5 million people have been infected with the SARS-CoV2 virus with approximately 650,000 (and rising) deaths. While primarily a respiratory virus, clinical observations appear to demonstrate nervous system involvement. These include those associated with the CNS (headache, confusion, seizure, stoke), PNS (pain, anosmia, ageusia) and enteric nervous systems (ENS, diarrhea). What is not fully understood- at this time- is how the SARS-CoV2 virus produces these nervous system disorders. We also do not know if the impact(s) on the nervous system will persist, or possibly even become apparent, post infection. Previous studies in my lab examining neurotropic (H5N1 influenza, western equine encephalitic virus) and non-neurotropic (pandemic H1N1 influenza) have shown that each can produce immediate and/or delayed effects in the CNS, including induction of pathologies seen in Parkinson?s disease. Related to SARs-CoV2, a number of recent studies, using autopsy material has examined the localization of SARS-CoV-2 virus in the brain. From these studies, approximately 36% had apparently low levels of viral SARS-CoV-2 RNA and protein in brain, although in each of these studies a complete cellular and localization map have not been reported. Also, it is not known if the viral particles found in the CNS were present intracellularly due to inherent neurotropism or were only present in the CNS due to breaches in the cerebral vasculature. (i.e., secondary to cerebrovascular damage). Even without neurotropism, an understanding of changes in the nervous system are critical since it is that any immediate and/or delayed effects may result from dysfunctional signals (peripheral cytokine storms) that arise outside of the nervous system; yet impact the function and, perhaps, survival of neurons. To address these unanswered questions, two specific aims are proposed. In Specific Aim 1, we will empirically determine the neurotropic potential of the SARS-CoV2 virus (USA-WA1) throughout its natural period of infection in the CNS, PNS and ENS in C57BL/6J mice and C57BL/6J mice expressing a human ACE2 receptor (K18-hACE2, B6.Cg-Tg(K18-ACE2)2Prlmn/J). We will also examine the induced inflammatory response in the periphery and brain. In Specific Aim 2, we will determine if resolved SARS- CoV2 infection can sensitize SNpc DA neurons to agents that have been shown to induce parkinsonism (paraquat and rotenone) in mice and humans as well as if it can exacerbate the spread and extent of alpha-synuclein pathology. These aims and associated experiments will allow us to directly determine the neurotropic and immunogenic potential of SARS-CoV2. They will also allow us to determine this virus has the potential to sensitize neurons to exogenous insults as has been demonstrated with some other respiratory viruses. Understanding if this pandemic virus affects the CNS and in particular, the basal ganglia is important for both short term treatment as well as longer-term management of post infection effects. Additionally, understanding the neuropathological sequalae of SARS-CoV2 on the nervous system will be necessary for later studies examining if a therapeutic intervention (i.e. vaccine or modulator of inflammatory response) can protect against primary and/or secondary nervous system effects of this respiratory infection.