|Investigators/Authors: J. Timothy Greenamyre, MD, PhD, University of Pittsburgh
Objective: To define the bidirectional interactions between the key PD-related protein, alpha-synuclein, and cellular ‘power plants’, mitochondria, in causing neurodegeneration in PD
Background: Impairment of the cellular ‘power plants’, mitochondria, appears to play an important role in many forms of PD. In addition, genetic mutations in a protein called alpha-synuclein can also cause rare forms of the disease. Moreover, individuals who simply make too much of the normal (non-mutated) alpha-synuclein protein are also at high risk of developing PD. We now have preliminary evidence that mitochondrial dysfunction and alpha-synuclein are closely intertwined:
- Mitochondrial impairment causes accumulation and aggregation of alpha-synuclein into ‘Lewy bodies’.
- Treatment of rat dopamine neurons with pre-formed fibrils of alpha-synuclein suppresses mitochondrial respiration and causes free radical formation.
- Genetically modified rats that express mutated alpha-synuclein are more sensitive to the damaging effects of mitochondrial dysfunction; they experience more degeneration of their dopamine system.
- Early studies suggest reducing the level of alpha-synuclein in dopamine neurons of living rats with ‘gene therapy’ protects them against mitochondrial impairment.
The overall goal of this proposal is to delineate the molecular mechanisms by which alpha-synuclein leads to mitochondrial impairment and by which mitochondrial impairment causes accumulation and aggregation of alpha-synuclein. Aim 5 will use a rat model of PD.
Methods/Design: Most of the work proposed here will use (in vitro) cultured dopamine neurons from the midbrain. Aims 1-3 will utilize a state-of-the-art 96-well Seahorse Biosciences XF96 respirometer. Aim 4 will use a Leica fluorescence live cell imaging system and an Olympus laser scanning confocal microscope.
Aim 1: Determine the time-course of the effects of alpha-synuclein on mitochondrial respiration in intact living dopamine neurons.
Aim 2: Determine the dose-response of the effects of alpha-synuclein on mitochondrial respiration in intact living dopamine neurons.
Aim 3: By permeabilizing the plasma membrane of intact living neurons, and supplying specific mitochondrial substrates (fuels for energy production), we will examine precisely where in the respiratory chain alpha-synuclein is exerting its effects.
Aim 4: Characterize the time-course and dose response of alpha-synuclein in enhancing mitochondrial free radical formation in intact living dopamine neurons. Effects in dopamine neurons will be compared to other types of neurons.
Aim 5: Determine whether viral-mediated ‘gene therapy’ to reduce the level of alpha-synuclein in dopamine neurons will protect against parkinsonism in an accurate rat model of PD.
Relevance to Parkinson’s disease: Both mitochondrial impairment and alterations alpha-synuclein may cause PD, and our recent work suggests these two seemingly disparate causes are, in fact, closely related. The work proposed here will not only help to define the relationship between these pathogenic processes, it will also determine whether it is a viable therapeutic target.
September 2013 Project Update:
Our findings tie together two seemingly unrelated disease mechanisms in PD - alpha-synuclein and mitochondrial impairment. This work has important therapeutic implications: If we can devise a means of minimizing the interaction of alpha-synuclein with TOM20, it should be possible to simultaneously (i) reduce the toxicity of alpha-synuclein and (ii) improve the function of mitochondria.
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Investigators/Authors: David G. Standaert, MD, Ph.D; Andrew West, PhD; Talene Yacoubian, MD, Ph.D.
Objective: To identify inflammatory signaling mechanisms in Parkinson disease (PD) which may provide targets for neuroprotective therapies.
Background: Inflammation of the brain, characterized by activated microglia, infiltration of immune cells from the periphery (T and B Cells) and production of potentially damaging cytokines, has long been recognized as a feature of Parkinson disease. In recent years, however, there has been recognition that this inflammation may occur early in the disease process, and may be a key driver of neurodegeneration. We have developed an animal model and in vitro systems, both based on the overexpression of human alpha-synuclein, which reproduce many of the key features of inflammation seen in human PD. We are using these models to explore the mechanisms of inflammation, and to identify potential targets for treatment.
Methods/Design: In this work, we will use a mouse model in which we induce overexpression of human alpha-synuclein using an AAV (adeno-associated viral) vector. In previous studies, we have found this induces robust neuroinflammation early, as soon as 2 weeks after injection, and causes loss of dopamine cells later, at 6 months after injection. We will also use a cell culture model in which we isolate microglia from mice at postnatal day 3-5, and stimulate them with aggregated forms of alpha-synuclein. There are several potential modulators of inflammation, but initially we plan to focus on the role of microRNA’s (miRNA’s). These are a class of small mRNA molecules that are increasingly recognized as having a critical role in controlling inflammatory states in a wide range of other disorders, but have not been examined closely in PD. We will explore changes in miRNA expression in both in vivo models, and correlate these with inflammatory processes.
Relevance to Diagnosis/Treatment of Parkinson Disease: These studies are intended to lay the groundwork for an understanding of the mechanisms which trigger and sustain inflammation in PD. The focus on miRNA’s is potentially important, because these signaling molecules are increasingly recognized as a potential target of therapies for a variety of different inflammatory disorders, although they have not been studied in PD. It is possible to design strategies to enhance or suppress specific miRNA’s in vivo, and an outcome of our work may be the development of a specific strategy that could be adapted to treatment of human PD.
It is also important to appreciate the broader significance of anti-inflammatory strategies in treating human PD. A variety of potential upstream causes of PD have been identified, which include both genetic factors (such as alpha-synuclein and LRRK2), chaperone modifiers (such as 14-3-3 proteins) and potential environmental triggers (such as exposures to pesticides). It is also known, however, that the degenerative process in PD begins long before the clinical symptoms are apparent. In patients already experiencing symptoms of PD, strategies which attempt to altering the upstream triggering factors may not be successful. It is quite clear, however, that inflammation is active at the time the symptoms appear, and remains active throughout the disease course. Thus, a treatment which reduces inflammation may be effective even after the disease is established, and may reduce or present progression regardless of the upstream trigger.
September 2013 Project Update:
In this work, we have used a mouse model in which we induce overexpression of human alpha-synuclein using an AAV (adeno-associated viral) vector. Using this model, we have observed an initial reduction of miR-124 expression at two weeks post-transduction followed by increased expression at four weeks; miR-155 expression is enhanced at two weeks. To test the importance of miR-155 in promoting the inflammatory response in this model, we examined the effect of alpha-synuclein activation in mice that are lacking miR-155. At four weeks post-transduction of AAV2-SYN, there was a marked decrease in the inflammatory response in these mice, compared to WT animals. This finding suggests that inhibition of miR-155 may protect against the loss of neurons by reduction of the inflammatory response. We are currently testing whether dopaminergic neuron loss is reduced in these mice lacking miR-155 at six months.
Because the degenerative process in PD begins long before the clinical symptoms are apparent, strategies which attempt to alter the upstream triggering factors may not be successful. It is quite clear, however, that inflammation is active at the time the symptoms appear, and remains active throughout the disease course. Thus, a treatment which reduces inflammation may be effective even after the disease is established, and may reduce or present progression regardless of the upstream trigger.
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