The Morris K. Udall Center of Excellence in Parkinson's Disease Research at Brigham and Women's Hospital, Harvard Medical School

Director: Peter T. Lansbury, Jr., Ph.D.

Title: Familial Parkinson’s Disease: Clues to Pathogenesis

Central Theme and Center Description

The overall approach of the Brigham Udall Center is to apply the study of the rare monogenic forms of Parkinson’s disease (PD) to uncover salient clues to the etiology of the more common sporadic forms of the disease.  The Brigham Udall Center has generated novel murine models of disease and employs neuronal cell culture, postmortem human brain tissue, yeast expression systems, and recombinant purified proteins in our complimentary and collaborative research efforts.  There has been a longstanding and program-wide interest in a-synuclein (aSyn).  More recent efforts have investigated the role of parkin in idiopathic and familial disease, structure-function studies of DJ-1, and a search for the physiological function of aSyn in neural cells.  Furthermore, integrated efforts across multiple labs are underway to understand the contribution of pathogenic mutations in LRRK2, the most common genetic cause of PD, to neuronal dysfunction.

 

Center Structure

Over the past several years the Brigham Udall Center has evolved into what is currently a complimentary program involving protein biochemistry and molecular biology to investigate LRRK2 structure and function (Gregory Petsko and Matthew LaVoie labs) and aSyn (Dennis Selkoe and Clemens Scherzer labs) with a vertebrate modeling effort in both areas (Jie Shen lab).  Dr. Peter Lansbury, Director of the Center, oversees the broad direction of the research program, and holds quarterly PI meetings where opportunities for collaboration, resource sharing, and strategy are openly discussed.

 

Recent Significant Advances

a-Synuclein

Increased a-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant PD in rare families. Variation in SNCA expression may be equally critical in sporadic, genetically complex PD.  By combining large-scale gene expression studies in humans with genetic complementation, gene silencing, and chromatin immunoprecipitation analyses in cells, The Scherzer and Schlossmacher labs found that GATA transcription factors directly regulate the expression of SNCA. GATA-1 induced a 62-fold increase in Snca mRNA and a 6.9-fold increase in aSyn protein. Silencing of endogenous neuronal GATA-2 induced a reproducible 28% decrease in SNCA mRNA and 46% decrease in aSyn in dopaminergic cells (Scherzer et al., 2008). Importantly, emerging data from a large population-based case-control association study indicate direct associations between PD and common sequence variants in both the GATA target aSyn intron-1 and the substantia nigra transcription factor gene GATA2 (Payami & Scherzer, in preparation). This GATA2-SNCA link can be exploited for understanding the role of aSyn production in PD, and suggests a novel path for developing therapeutics designed to lower aSyn production.

To date, the only specific biochemical function that has been proposed for aSyn is the inhibition of phospholipase D (PLD), which was supported by data from multiple groups. Through intensive efforts using multiple experimental systems and in collaboration with experts on both PLD and aSyn and across Udall Centers, The Selkoe lab has shown that aSyn is not likely to inhibit PLD (Rappley et al., 2009a), and therefore that PLD inhibition is not likely to contribute to PD pathogenesis.  

Consistent with the potential importance of aSyn within the lipid environment, several reports have suggested a link between aSyn expression and phospholipid membrane composition.  Therefore, we examined the phospholipid composition in the brains of aSyn transgenic or knockout mice.  Using a novel phospholipid mass spectrometry technique, we found significant differences in phospholipid composition across mouse strains, genders, and ages.  While we did find some differences across aSyn genotypes, these were less dramatic than the aging- and gender-related findings (Rappley et al., 2009b).  Given the relatively modest effects of aSyn on lipid composition, further work will be required to determine whether the function of aSyn may be affected by aging-related lipid changes in the brain, thus contributing to PD pathogenesis.

Parkin

The LaVoie lab has recently shown that parkin prevents cytochrome c release and downstream apoptosis, an effect that can be readily observed from isolated mitochondria in a cell-free system (Berger et al., 2009).  Importantly, this effect was observed with endogenous parkin, and could not be replicated with PD-linked pathogenic parkin mutants.  We found that exogenously applied parkin could associate with isolated mitochondria but that binding per se was not sufficient to reduce cytochrome c release.  Rather, parkin must be expressed within the cytoplasm of the living cell in order to alter mitochondrial cytochrome c release.  These data may provide important insight into the localization of the specific parkin substrates involved in its protective function within neuronal cells, as well as the mechanisms involved.

LRRK2

Mutations in LRRK2 are the most common genetic cause of familial PD, and may also contribute to the pathogenesis of idiopathic PD.  In particular, three distinct mutations have been identified at position R1441. The Shen lab has recently characterized a novel mouse mode of familial PD by generating a knock in (KI) mouse in which the R1441C mutant form of LRRK2 is expressed under the control of the endogenous regulatory elements. While 1441C KI mice do not develop dopaminergic neurodegeneration or exhibit alterations in total striatal dopamine, they show reductions in amphetamine-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells (Tong et al., PNAS 2009). The introduction of the R1441C mutation also impairs dopamine D2 autoreceptor-mediated functions, as indicated by decreased responses of KI mice in locomotor activity to the inhibitory effect of a D2 receptor agonist, quinpirole.  Furthermore, the firing of nigral neurons in R1441C KI mice also displays reduced sensitivity to suppression induced by quinpirole, dopamine or amphetamine. Thus, the R1441C mutation in LRRK2 impairs stimulated dopamine neurotransmission and dopamine D2 autoreceptor-mediated functions, which may represent pathogenic events which precede dopaminergic degeneration in PD brains. We believe that our study represents a major advance in understanding LRRK2 dysfunction in the pathogenesis of PD and provides further supporting evidence for presynaptic dopaminergic dysfunction being a pathogenic precursor.

 

Available Resources

Tissue and Genomic Depository and Analyses

The lack of biological markers that track disease progression is a critical roadblock in the development of disease-modifying therapeutics for PD.To accelerate the discovery and validation of molecular diagnostics that track progression of early-stage PD and AD we are conducting the Harvard NeuroDiscovery Center Biomarker Study (HBS) (Co-Directors: Drs. Scherzer, Hyman, and Ivinson). HBS is a Harvard-wide, longitudinal, case-control study of 2,000 individuals with early-stage PD, MCI/AD, and controls without neurologic disease funded by the Harvard NeuroDiscovery Center and the NIH. Biological and clinical phenotypes are tracked at three visits over a two-year period. Over 1,000 of 2,000 subjects have been enrolled and over 126 have completed three visits. 25,000 biospecimens have been processed, quality-controlled, barcoded and stored ready for analysis. Nine biomarker studies nested in HBS examining analysts ranging from metabolites, to proteins, to mRNAs, to DNA are currently active.This unique longitudinal biobank with detailed linked clinical data will greatly accelerate biomarker development by shrinking study time (from five years to assay run-time) and costs (from multi-million dollar costs to assay costs).

Mouse models available (Shen lab)

• Parkin germline knockout, DJ-1 germline knockout, PINK-1 germline knockout, LRRK2 germline knockout, LRRK2 R1441C knockin
• Behavioral analysis suite

Molecular Biology/Biochemistry Reagents

• Expression plasmids for WT and PD-linked mutant proteins, as well as shRNA-based silencing constructs for: LRRK2, parkin, PINK1, DJ-1, aSyn (Lansbury, LaVoie and Selkoe labs)
• Purified recombinant LRRK2 fragments, and full length WT and mutant isoforms of DJ-1 and aSyn (Petsko, Lansbury, Ray labs)
• Established RT-PCR for numerous PD-related genes (Scherzer and LaVoie labs)

Cell Culture Systems

• Cultured primary rat and mouse neurons (Selkoe and LaVoie labs) and numerous cell lines expressing (or silencing) most PD-related gene products (LaVoie and Selkoe labs)

 

Plans for the Coming Year

As the structure of the Brigham Udall Center has focused on biophysical, biochemical, molecular, and behavioral efforts to examine the roles of LRRK2 and aSyn in the pathogenesis of PD, there are multiple avenues along which we will expand our work in the coming year.

Structural and functional consequences of LRRK2 mutations

The domain organization of LRRK2 suggests that there are at least two enzymatic functions in the protein. The first is a serine/threonine kinase function; mutations such as G2019S and I2020T can be considered direct gain of function as they lie on the catalytic loop of the kinase. However, the majority of our focus will be on the Roc domain, which stands out as a separate monophyletic group of the Ras-related superfamily of small GTPases. Recent crystal structure suggests that the Roc-GTPase is a domain-swapped dimer with the R1441 residue lying at the dimer interface. Importantly, mutations at this site (R1441C/G/H) are associated with familial PD.  We have cloned and purified this domain and have shown in a preliminary experiment using thermofluor^® technology that R1441G is a highly destabilizing mutation that has severe thermodynamic consequences on protein dimerization. Modeling studies also suggest that Y1699C in the COR domain may be a dimer-disrupting mutation.  Ongoing work with recombinant proteins will be further translated into intact cells and primary neurons to explore the importance of dimer-formation to LRRK2 activity and complex assembly.  With R1441C knockin mice already generated (Tong et al., 2009), there will also be complimentary work conducted in this novel animal model.  In addition, further behavioral and molecular characterization of the LRRK2 knockin animals will likewise proceed. 

Regulation and physiological role of aSyn

We will characterize GATA2-controlled expression of endogenous aSyn.  Furthermore, the impact of GATA2 regulation on disease-linked aSyn oligomers will be examined in dopamine neurons using a variety of cell lines and assays.  We will also determine whether soluble, cytoplasmic aSyn oligomers disrupt mitochondrial calcium homeostasis and impair mitochondrial motility in neural cell culture systems.

 

Select Recent Publications

Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J, McGoldrick M, Mollenhauer B, Bresnick EH, Schlossmacher MG. GATA transcription factors directly regulate the Parkinson's disease linked gene alpha- synuclein. Proc Natl Acad Sci U S A. 2008, 105 (31):10907-12.

Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, Yun HJ, Kim CH, Han BS, Tong Y, Shen J, Hatano T, Hattori N, Kim KS, Chang S, Seol W. LRRK2 regulates synaptic vesicle endocytosis. Exp Cell Res. 2008, 314 (10):2055-65.

Rappley I, Gitler AD, Selvy PE, LaVoie MJ, Levy BD, Brown HA, Lindquist S, Selkoe DJ.  Evidence that a-synuclein does not inhibit phospholipase D.  Biochemistry  2009a, 48 (5):1077-83.

Vamvaca K, Volles MJ, Lansbury PT. The first N-terminal amino acids of alpha synuclein are essential for a helical structure formation in vitro and membrane binding in yest. J Mol Biol. 2009, 389(2):413-24.

Rappley I, Myers DS, Milne SB, Ivanova PT, LaVoie MJ, Alex Brown H, Selkoe DJ.  Lipidomic profiling in mouse brain reveals differences between ages and genders, with smaller changes associated with a-synuclein genotype.  J Neurochem  2009b, in press.

Liu Z, Meray RK, Grammatopoulos TN, Fredenburg RA, Cookson MR, Liu Y, Logan T, Lansbury PT.  Membrane associated farnesylated UCH-L1 promotes alpha synuclein neurotoxicity and is a therapeutic target for Parkinson's disease. Proc Natl Acad Sci U S A. 2009, 106 (12):4635-40.

Tong Y, Pisani A, Martella G, Karouani M, Yamaguchi H, Pothos EN, Shen J. R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice. Proc Natl Acad Sci U S A. 2009, in press.

Berger AK, Cortese GP, Amodeo KD, Weihofen A, Letai AG, LaVoie MJ.  Parkin selectively alters the intrinsic threshold for mitochondrial cytochrome C release.  Hum Mol Genet  2009, in press.

 

Public Health Statement

The molecular substrates of PD pathogenesis remain unclear despite decades of intense efforts by numerous talented laboratories.  Our hope is that by focusing attention towards two of the most well characterized genetic causes of PD with an integrated research program incorporating recognized expertise in protein chemistry, cell biology, and vertebrate animal modeling we will uncover critical biochemical pathways relevant to all PD cases.  This information would then be available to better inform the development of novel therapeutic interventions at our Laboratory for Drug Discovery in Neurodegeneration facility and elsewhere.