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7. The Parkinson’s Institute is using its distribution to fund the following:
Grant to: Seung-Jae Lee, PhD
Project Title: The Folding and Aggregation of Alpha-Synuclein in Complex Systems
This research, initially funded through a grant from The Abramson Family Foundation, has been extremely successful, producing a number of publications in significant scientific journals such as The Journal of Biological Chemistry.
It is Dr. Lee's hope that at the end of this research project, he will move the scientific community that many more steps into understanding the way the origin and the way in which Parkinson's disease develops in the human through his study of alpha-synuclein.
The function and survival of neurons relies on the action of numerous proteins. Each protein has a unique conformation (folding) for its function. Various environmental insults and genetic defects can lead certain proteins to abnormal conformations, accumulation of which could be deadly to neuronal cells.
One of the leading hypotheses to explain the origin and development of Parkinson's disease (PD) is that the accumulation of abnormally folded proteins causes neuronal dysfunction and death. Normal neurons have "quality control systems" that recognize and remove the abnormally folded proteins. However, these misfolded proteins can accumulate when the damage to the proteins exceeds the cellular quality control capacity or when there are defects in the quality control system itself.
The goal of this research program is (1) to understand the molecular mechanism of protein misfolding and what triggers the misfolding, (2) to elucidate the mechanism of misfolded protein clearance, and (3) to understand how accumulated misfolded proteins cause neuronal dysfunction and death. This study will significantly enhance our understanding on the fundamental mechanism underlying the process of development in PD and related neurodegenerative diseases at the molecular level, and lead to the identification of therapeutic targets. Furthermore, this study will provide the scientific foundation for developing better animal models and drug screening systems. The results of this study will lead to better and new strategies to possibly prevent PD and/or slow its progression.
December 2004 Project Update:
To study the effects of a-synuclein aggregates on other components of the mitochondrial system, we examined the distribution of the mitochondrial motor proteins. The mitochondrial system uses two types of molecular motors depending on the direction of movement: the plus-end directed motors and the minus-end directed motors transport cargo towards the plus-end and the minus-end of the mitochondrial's, respectively (Goldstein and Yang, 2000). We have analyzed the distribution of two plus-end motor components, kinesin heavy chain (KHC) and KIF3A, and two minus-end motor components p150glued and p50/dynamitin. We have found that only the sub units co-localize with a-synuclein aggregates, while the plus-end motor subunits are almost completely excluded from the aggregates. It has been suggested that protein aggregates are transported by minus-end directed motors to form a higher-order inclusion bodies (Kopito, 2000). Our study confirms that a-synuclein aggregates are handled by a specific motor system, and thus may lead to its impairment when the system is loaded beyond its capacity.
Trafficking defects in cells with a-synuclein aggregates
To further characterize the effects of microtubule dysfunction in a-synuclein overexpressing cells, we investigated the effects of a-synuclein overexpression on the microtubule-dependant trafficking. The temperature-sensitive mutant form of the vesicular stomatitis virus G protein (VSV-G) is a widely-used model protein in studying trafficking through biosynthetic pathway (Pepperkok) et al., 1993; Scales et at. 1997; Seemann et al., 2000). Here, we studied the trafficking property of this protein using an adenoviral vector bearing the GFP-tagged VSV-G gene. VSV-G failed to fold properly and accumulates in ER at the restrictive temperature of 39.5C (t=0). But after the cells are transferred to the permissive temperature of 31.5C, VSV-G folds rapidly and is transported progressively from ER through the GA (t=20 min) to the plasmas membrane (PM; t = 60 min) in a time dependent manner.
To examine the effects of a-synuclein aggregation on VSV-G trafficking, we performed an experiment in which distribution of VSVG-GFP was visually observed. VSVG-GFP was expressed in COS-7 cells overexpressing a-synuclein at a restrictive temperature for 6.5 hours, and then at a permissive temperature for 60 min, after which the cells were fixed, stained with a-synuclein anti-body, and examined with confocal microscopy. The cells with diffuse a-synuclein expression without apparent aggregation often show typical VSV-G distribution; intense Golgi localization with clear cell surface expression. On the other hand, the cells with no noticeable spherical a-synuclein aggregates tend to show little VSV-G protein on the cell surface and dispersed cytoplasmic distribution. This suggests that the trafficking of VSV-G may be impaired in cells with a-synuclein aggregates, thus supporting the conclusion that a-synuclein aggregation causes microtubule dysfunction.
Key Research Findings:
- Biochemical fractionation method to separate the a-synuclein aggregate subspecies from the cytoplasm has been established.
- The fractionation and EM studies show that the juxtanuclear inclusion bodies are filled with a-synuclein fibrils and that at least two different prefibrillar spherical aggregates are formed in the course of fibrillation
- Time-course and microtubule-disruption experiments show that spherical aggregates are the precursors for fibrillar inclusion bodies.
- a-Synuclein fibrillation in cells is tightly coordinated with the microtubule-dependant inclusion-forming process.
- Formation of prefibrillar oligomeric a-synuclein aggregates is associated with Golgi fragmentation, suggesting the prefibrillar intermediates as being pathogenic species.
- Fibrillar inclusion bodies seem to be a consequence of cellular effort to remove toxic protein aggregates and damaged organelles from cytoplasm.
- Deleterious effects of a-synuclein aggregation on Golgi fragmentation and cell viability were confirmed in post-mitotic neuronal cells.
- a-Synuclein aggregation causes the disruption of the microtubule network and the intracellular trafficking, leading to Golgi fragmentation and neuritic degeneration.
Conclusions
A growing body of evidence suggests that aggregation of a-synuclein might be the fundamental cause of many neurodegenerative diseases. Several groups have developed cell culture models to study the cytotoxic effect of a-synuclein, and some of them indeed have observed enhanced cell death when a-synuclein, especially its mutant forms, was overexpressed (Ostrerova et al., 1999; Saha et al., Iwata et al., 2001; Zhou et al., 2002). However, the link between a-synuclein aggregation and cell death has not been clearly addressed in these model systems, nor are the molecular mechanisms underlying the toxicity known. We have begun to address these issues in a COS-7 cell model, and found that a-synuclein aggregation/oligomerization is tightly associated with Golgi fragmentation and cell death (Gosavi et al., 2002). More recently, fragmentation of the GA has been confirmed in a neuronal cell model, and the mode of Golgi fragmentation appears identical to that caused by microtubule-disrupting agents; dispersed Golgi fragments are localized to the transitional endoplasmic reticulum. Indeed, we found that a-synuclein aggregation led to the disruption of the microtubule network in neuronal models. Microtubule disruption then caused intracellular trafficking impairment and neurite degeneration. These data identify microtubule dysfunction as being the link between a-synuclein aggregation and neurodegeneration.
Recent studies show that the microtubule transport system also plays a role in inclusion body formation, as part of the cellular response to the aggregation of misfolded proteins (Johnson et al., 1998 Garcia-Mata et al., 1999; Johnson et al., 2000). Aggregation of proteins could occur anywhere in the cytoplasm, resulting in many small aggregate particles scattered throughout the cell. These particles are deposited in the pericentriolar region, adjacent to the microtubule-organizing center, by retrograde transport on microtubules. These microtubules-dependent deposits of aggregates are called aggresomes (Johnson et at. 1998) and may explain the biogenesis of inclusion bodies found in neurological diseases, such as Lewy bodies in PD. The microtubule-dependent nature of inclusion formation suggests that extensive protein aggregation in neuron may place a tremendous burden on the mircrotuble-dependent nature of inclusion formation suggests that extensive protein aggregation in neurons may place a tremendous burden on the microtubule transport system, causing it to malfunction. Small a-synuclein oligomers are transported on the microtubules and deposit to form inclusion bodies (Lee and Lee, 2002). In addition, our current data indicate that a-synuclein overproduction causes the disruption of neuronal mitochondrial network, probably via aggregation. This, in turn, leads to downstream degenerative changes, such as neuritic degeneration and Golgi fragmentation, which are common pathological features shared by many human neurodegenerative diseases (Gonatas et al., 1998). Determining the precise mechanism by which abnormal a-synuclein disturbs mitochondrial integrity will provide insight into the early pathogenic mechanism of PD and other synucleinopathies.
The Parkinson's Institute is also joining the National Parkinson Foundation in funding the following:
Grant to: Dr. Lee He-Jin
Project Title: Role of autophagic lysosomal pathway in the degradation of alpha synuclein aggregates.
The protein alpha synuclein is important for the integrity and normal functioning of the dopamine cells in the substantia nigra. It is believed that if molecules of alpha synuclein are mis-shapen can become toxic, poisonous, to the cell. Such mis-shapen molecules may be removed by a "clearing" system in the cell that centers on a group of particles called lysosomes. It is believed that if this "clearing" system isn’t working properly then mis-shapen aggregates of alpha syncuclein accumulate and "poison" the cell. This study will look at the ability of the lysosomes to clear such mis-shapen aggregates of alpha synuclein in the tan.
November 2004 Project Update:
Dr. He-Jin Lee and her colleagues at the Parkinson's Institute (Sunnyvale, California) have created a human neuronal tumor cell line that they have utilized in testing endogenous and exogenous (naturally-occurring and additive) dopamine (DA) as it affects and is affected by the protein alpha-synuclein (@-synuclein). This protein, when mutated, has been linked to certain familial parkinsonisms and has also been found in brains of deceased patients with classic PD in an aggregated or clustered form commonly known as Lewy bodies, long considered a hallmark of the disorder.
The cell line can be differentiated into cells that act like neurons that produce DA, making it possible to measure @-synuclein in the outer parts of the cell called the cytosol, showing that the protein increases as the amounts of DA increase. Future work by this group will attempt to delineate the details of how this aggregation affects the functions of the DA-containing cells in the hope that clues will be found leading to a better understanding of the pathogenesis of PD.
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