Phyllis L. Faust, MD PhD - US grants

DESCRIPTION (Verbatim from the Applicant's Abstract): Zellweger syndrome is a human peroxisomal biogenesis disorder that results in abnormal neuronal migrations in the CNS and severe neurologic dysfunction. The principal investigator has developed a murine model for this disorder by targeted deletion of the PEX2 peroxisomal gene and has demonstrated the presence of peroxisomal defects and a cerebral cortical abnormality in newborn mice. PEX2-deficient mice that survive in the postnatal period also develop severe cerebellar abnormalities. In an inbred strain, PEX2 mice appear to develop extensive neuronal lipidosis, with a prominent concentration in the inferior olivary nucleus. These mice provide an important animal model to understand the role of peroxisomal function during CNS development. The principal investigator proposes an integrated series of cellular and molecular studies to begin to answer the following questions. 1. What is the effect of the peroxisomal deficiency on neuronal proliferation, migration, survival, and differentiation during murine CNS development? Neuronal precursor cells will be labeled in vivo with mitotic markers or retroviral infection and their proliferative rate, subsequent migration and final differentiation examined. The investigator will directly visualize the dynamics of migrating neurons in vitro by video microscopy using cultured brain slices. The architectural changes that occur in the mutant brains will be examined with cell type specific markers having established developmental patterns to define the onset and the evolution of the peroxisomal disease process. Ultrastructural studies will define further the cell type specific peroxisomal pathology observed at various stages of development. The effect of peroxisomal deficiency on neuronal survival and postmigratory differentiation will be examined. 2. What is the role of cell autonomous function versus epigenetic factors in causing the developmental defects? Two major strategies will be used to approach this question and include: a. in vivo transplantation of normal or peroxisome-deficient neuronal progenitors into normal or mutant developing cortex. These studies will evaluate whether the cellular defects observed in the brain of peroxisome deficient neurons are intrinsic to the neurons versus secondary to defects in other brain cells and/or environmental alterations; and b. liver/gut specific PEX2 transgene expression to correct hepatic peroxisomal abnormalities and evaluate the role of circulating toxic factors and/or malnutritions that result from hepatic dysfunction versus intrinsic brain metabolism in causing the CNS defects.

DESCRIPTION (provided by applicant): Our laboratory has been studying a murine PEX2 null animal model for the human peroxisomal biogenesis disorder Zellweger syndrome. We have determined that peroxisomal dysfunction in vivo affects neuronal proliferation, migration, survival and differentiation during CNS development. However, the contributions from intrinsic CNS defects versus extrinsic systemic organ dysfunctions and the role of peroxisomal lipid deficiencies or specific peroxisomal pathway defects have not been determined. We propose an integrated series of in vitro and in vivo approaches to address the following questions: Specific Aim #1. Are the defects in neuronal migration and differentiation in PEX2 -/- mice caused by factors intrinsic or extrinsic to the CNS? What is the role of peroxisomal lipid deficiencies or specific defects in peroxisomal B-oxidation or plasmalogen pathways in causing these abnormalities? We will directly visualize the dynamics of migrating neurons from control and PEX2 -/-mice by videomicroscopy using cerebral and cerebellar slice cultures and cerebellar neuron-glial cultures. The in vitro differentiation of control and PEX2 mutant cerebellar Purkinje cells and granule neurons will be examined. We will determine the effect of correcting deficiencies for plasmalogens or docosahexaenolc acid on the cellular defects in PEX2 neurons. We will compare the CNS defects observed in PEX2 -/- mice with those seen in mice with peroxisomal defects limited to plasmalogen and/or B-oxidation pathways (MFE2 -/-, MFE2/MFE1 -/-, PEX7 -/-) using these assays for neuronal migration and differentiation. We will evaluate the role of CNS intrinsic versus systemic peroxisomal dysfunction in the pathogenesis of CNS defects by both in vitro and in vivo transfection methods to restore PEX2 function in selected CNS cell types or brain regions. Alternatively, we will use in vivo transplantation of normal or peroxisome-defective neuronal progenitors into normal or mutant developing cerebral cortex or cerebellum. Specific Aim #2: Do hepatic factors contribute to the CNS defects in peroxisome defective mice? We will evaluate how CNS development is altered when bile acid deficiency is corrected in vivo in PEX2 -/-mice. We will determine whether bile acid products accumulate in the brain of PEX2 / mice. We will examine whether toxic substances from the PEX2 mutant liver cause CNS dysfunction by determining the effect of adding exogenous bile acid intermediates or hepatocyte conditioned medium on the in vitro neuronal defects in control, PEX2 -/, plasmalogen or B-oxidation defective mice. Specific Aim #3: What is the role for peroxisomes in developing cerebellar Purkinje cells and/or granule neurons? Conditional deletion of the PEX2 gene in cerebellar Purkinje cells and/or granule neurons will determine the role of peroxisomes for the development of these neurons in the absence of systemic organ peroxisome deficiency.

DESCRIPTION (provided by applicant): Essential tremor (ET) is among the most common neurological diseases, occurring in 4% of adults age 40 and older, and even more frequently in advanced age (i.e., 7-8% by age 70 and >20% by age 90+). Despite its high prevalence, the underlying pathogenesis of ET is poorly understood, and, as a consequence, current treatments are empiric and have poor efficacy. Human postmortem studies are currently the most robust avenue for advancing the study of the underlying pathophysiological mechanisms of ET, as genes have yet to be identified for ET, and no transgenic mice currently exist to provide an animal model. Our group has been conducting systematic postmortem studies, which have revealed identifiable structural changes in the brains of ET cases. We have demonstrated that neuropathologic findings in the vast majority of ET cases (>90%) localize to the cerebellum itself and, in particular, to the Purkinje cells (PC), which provide the entire neuronal output from cerebellar cortex. In ET brains, there is a 6- to 7-fold increase in damaged PC axons, identified as rounded swellings of the proximal portion of the PC axon (i.e., torpedoes). Strongly correlating with this PC axonal damage is an approximate 30-40% reduction in the number of PCs and an increase in the number of heterotopic (displaced) PCs. Our ongoing pathologic studies further indicates that the torpedo is likely only a marker of advanced PC axonal damage. Earlier changes are now becoming evident in ET brains, including a graded increase in small to intermediate-sized thickenings of the proximal PC axon (i.e., presumed precursor- torpedoes) and increases in PC recurrent axon collateral formation with increased sprouting along the intracortical segment of PC axons. These studies implicate PC degeneration as a core feature of the disease process in ET, associated with slowly progressive axonal damage and significant remodeling of intracortical cerebellar connectivity. While these postmortem studies have advanced our understanding of disease pathogenesis to a cellular level, it is time to now proceed to a molecular understanding. In this proposal, we will address whether molecular alterations can be identified in cerebellar PCs from ET patients vs. neurologically normal controls. We will perform gene expression analyses by microarray screening on RNA isolated from PCs of ET versus control autopsy brains. We will employ laser-capture microdissection to specifically target PCs, thereby facilitating a precise evaluation of cell-specific changes associated with ET. ET brains will be limited to those with an absence of other neurodegenerative pathologies at autopsy. Analysis of the resultant expression data will identify genes and/or molecular pathways that are differentially expressed and/or biologically grouped in ET versus control PCs. Changes in gene expression will be validated by quantitative real time PCR. This study will be the first to initiate a molecular expression approach in ET research, and will provide a basis for proposing molecular causal mechanisms for ET that will be the focus of future investigations.

DESCRIPTION (provided by applicant): Despite its high prevalence, essential tremor (ET) it is among the least-studied and most poorly- understood neurological diseases. On the most basic biological level, little is known about its underlying pathologic-anatomy and pathophysiology. Recent postmortem studies report a 30 - 40% loss of Purkinje cells (PCs) in ET, suggesting that on a mechanistic level, this common neurological disease could be neurodegenerative. But postmortem results have been mixed. At the moment, the central debate in the ET field revolves around the question, is PC loss a feature of ET? Unfortunately, the approach to this question has thus far been limited to postmortem studies and a fresh approach is needed. PCs, in the cerebellar cortex, are the major storehouse of brain gamma-aminobutyric acid (GABA), releasing their GABA into the synaptic cleft at the level of the cerebellar dentate nucleus. Thus, cerebellar dentate GABA level is a convenient in vivo marker of PC number. Furthermore, N-acetylaspartate (NAA), an amino acid found in the cytosol of neurons, is a convenient in vivo marker of neuronal integrity in the cerebellar cortex. The long-term goal of the proposed research is to elucidate the basic nature of the underlying pathophysiology of ET. The objective of this research project, which is the next step toward attainment of this long-term goal, is to perform in-vivo magnetic resonance spectroscopy (MRS) to quantify levels of GABA in the cerebellar dentate and NAA in the cerebellar cortex in 50 ET cases vs. 50 controls, both cross-sectionally and longitudinally. The central hypothesis for the proposed research is that there is a progressive destruction of PCs in ET. We plan to accomplish the overall objective of this application by pursuing the following three aims. Aim 1: In this cross-sectional neuroimaging aim, we will use MRS at baseline (Years 1 - 2) to assess in vivo cerebellar dentate GABA levels and cerebellar cortex NAA levels (NAA/total creatine, tCR) in ET cases and controls. Aim 2: In this longitudinal neuroimaging aim, we will perform a second MRS study (Years 4 - 5) to assess in vivo cerebellar cortex NAA/tCR levels and, in an exploratory manner, dentate GABA levels, to determine whether there is a longitudinal decline in these levels in 50 ET cases in excess of any longitudinal decline in these levels in 50 controls. Aim 3: We will cross validate the in vivo MRS findings with postmortem histological and biochemical findings in the brains of 10 - 15 of 50 ET cases whom we expect to die during the 5 year study. We expect that the proposed research will elucidate the underlying disease pathophysiology and provide useful diagnostic biomarkers as well as markers of disease progression for future neuroprotective trials.

? DESCRIPTION (provided by applicant): Over the past 5 - 8 years, we have identified a cluster of morphological changes in the essential tremor (ET) cerebellum, predominantly centered in/around the Purkinje cell (PC). The discovery of ET-related pathology has generated great interest but it has also raised difficult questions. Several of these pathologies have also been observed in primary cerebellar neurodegenerative diseases such as spinocerebellar ataxias (SCAs) and multiple system atrophy (MSA), but the degree to which these changes occur has not been formally studied or compared with that in ET. Interestingly, the cerebellum is now increasingly being implicated in tremor generation in other diseases such as Parkinson's disease (PD) and dystonia, yet their cerebellar pathology is presently unexplored. Hence, there is a large morphologic data gap. On a more primary level, we recently performed laser-capture microdissection (LCM) to specifically target PCs, thereby facilitating a precise evaluation of cell specific molecular changes in ET. We obtained a highly novel differential gene expression profile by direct sequencing of RNA (RNA-seq) isolated from PCs of ET vs. control brains. We identified 47 differentially expressed transcripts, which code for proteins that regulate neuronal function. However, a parallel set of LCM-RNA-seq studies, exploring the molecular biology of PCs in PD, dystonia, MSA and SCA, have yet to be performed. This represents a second, large molecular, data gap. This five-year proposal, which uses postmortem tissue from patients with ET well as from patients with a range of other cerebellar disorders, has two aims. Specific Aim 1: To undertake detailed postmortem studies of the cerebellum, comparing morphological changes in the cerebellum of ET patients to those of patients with primary cerebellar degenerative diseases (SCA and MSA) as well as those of patients with neurological diseases with tremor and cerebellar involvement (PD and dystonia). We will assemble an initial discovery sample of 160 brains (50 ET, 25 SCA, 15 MSA, 30 PD, 15 dystonia, 25 controls) as well as a replicate sample of 160 brains, assessing pathological changes across several cerebellar compartments. Hierarchical cluster analysis of quantified variables will be used to determine whether there is a definable ET cluster as well as definable clusters for each of these other four diseases. Specific Aim 2: To explore the molecular biology of PCs across neurodegenerative diseases characterized by cerebellar involvement and/or tremor. Using a novel LCM-RNA-seq approach, we will determine whether molecular expression changes in PCs are the same or differ across these diseases. For these novel molecular studies, we propose to use 60 brains (10 each of ET, SCA, MSA, PD, dystonia, controls).

Over the past 5 - 8 years, we have identified a cluster of morphological changes in the essential tremor (ET) cerebellum, predominantly centered in/around the Purkinje cell (PC). The discovery of ET-related pathology has generated great interest but it has also raised difficult questions. Several of these pathologies have also been observed in primary cerebellar neurodegenerative diseases such as spinocerebellar ataxias (SCAs) and multiple system atrophy (MSA), but the degree to which these changes occur has not been formally studied or compared with that in ET. Interestingly, the cerebellum is now increasingly being implicated in tremor generation in other diseases such as Parkinson's disease (PD) and dystonia, yet their cerebellar pathology is presently unexplored. Hence, there is a large morphologic data gap. On a more primary level, we recently performed laser-capture microdissection (LCM) to specifically target PCs, thereby facilitating a precise evaluation of cell-specific molecular changes in ET. We obtained a highly novel differential gene expression profile by direct sequencing of RNA (RNA-seq) isolated from PCs of ET vs. control brains. We identified 47 differentially expressed transcripts, which code for proteins that regulate neuronal function. However, a parallel set of LCM-RNA-seq studies, exploring the molecular biology of PCs in PD, dystonia, MSA and SCA, have yet to be performed. This represents a second, large molecular, data gap. This five-year proposal, which uses postmortem tissue from patients with ET well as from patients with a range of other cerebellar disorders, has two aims. Specific Aim 1: To undertake detailed postmortem studies of the cerebellum, comparing morphological changes in the cerebellum of ET patients to those of patients with primary cerebellar degenerative diseases (SCA and MSA) as well as those of patients with neurological diseases with tremor and cerebellar involvement (PD and dystonia). We will assemble an initial discovery sample of 160 brains (50 ET, 25 SCA, 15 MSA, 30 PD, 15 dystonia, 25 controls) as well as a replicate sample of 160 brains, assessing pathological changes across several cerebellar compartments. Hierarchical cluster analysis of quantified variables will be used to determine whether there is a definable ?ET cluster? as well as definable clusters for each of these other four diseases. Specific Aim 2: To explore the molecular biology of PCs across neurodegenerative diseases characterized by cerebellar involvement and/or tremor. Using a novel LCM-RNA-seq approach, we will determine whether molecular expression changes in PCs are the same or differ across these diseases. For these novel molecular studies, we propose to use 60 brains (10 each of ET, SCA, MSA, PD, dystonia, controls).