Grants & Funding > The Parkinson Alliance Research Grants 2016

The Parkinson Alliance
is using its 2016 distribution to fund:

Does Air Pollution Increase the Risk of Parkinson’s Disease (PD)?

PROJECT TITLE:  Does Air Pollution Increase the Risk of Parkinson’s Disease (PD)?

Investigator/Author:  Jeff Bronstein MD, PhD

Objective:  The causes of PD are largely unknown.  Genetic factors appear to contribute approximately 20% to risk of developing the disease leaving environmental factors likely contributing the majority of risk.  A few environmental factors such as pesticides and head trauma almost certainly contribute to some individuals’ disease, these cannot explain the majority of risk that has yet been accounted for.  A recent study in Denmark found those exposed to high air pollution were at increased risk of developing PD and could account for a large percentage of the PD patients.  We have exciting preliminary data that support this theory.  Our objective in this proposal is to determine the biological plausibility of air pollution as a risk factor for PD and how it might cause it.

Methods/Design:  Diesel exhaust particles (DEP) is the major toxic constituent in air pollution and we will use an extract of DEP in our studies.  Zebrafish offer several advantages to other animal models including the fact that they are transparent and easily modified genetically.  Since alpha-synuclein accumulates in neurons in PD, we will test the effect of DEP on expression of alpha- synuclein using quantitative PCR and immunoassays.  We will also determine the effect of DEP on other proposed pathogenic pathways such as inhibition of protein breakdown, inflammation through microglia, and mitochondrial dysfunction.

Relevance to Treatment of Parkinson’s Disease:  There are still no treatments that slow or stop the progression of PD despite several attempts.  One reason for this is that we still do not know the causes of PD which is necessary to target specific pathways.  Based on extrapolation from the Danish study, more than half of all PD cases in Los Angeles could be caused by air pollution.  If this is found to be accurate, we will have identified new therapeutic targets.

Expected Outcome:  We anticipate that air pollution increases the risk of developing PD by more than one mechanism.   We have already found that DEP causes dopaminergic neuron loss in zebrafish and preliminary studies suggest that it increases alpha-synuclein expression.  Inflammation via microglia also likely contributes to the development of PD.  We will also determine if air pollution’s toxicity is dependent on alpha-synuclein since many disease modifying therapies are directed at lowering alpha-synuclein toxicity.

September 2018 Project Update:

We have continued to make progress in determining how air pollution increases the risk of PD.  The studies measuring the effects of diesel exhaust extracts on synuclein expression have been completed.  The results presented last year have been confirmed and are contained in Lisa Barnhill’s PhD dissertation and will soon be submitted for publication.  We have also fully characterized the transgenic zebrafish line we developed that allows us to measure the breakdown of proteins (including synuclein) by a process called autophagy.  Autophagy is a dynamic process and after finding all available tools were not sufficient to measure this, we created the GFP-LC3 zebrafish line and a novel method to measure to flux of autophagic vesicles.  A manuscript describing this is in press in the prestigious journal, Autophagy.  Using the GFP-LC3 zebrafish, we found that diesel exhaust impairs autophagic flux.  Furthermore, nilotinib which increases flux, protected the dopamine neurons from diesel exhaust toxicity indicating that this is one mechanism by which air pollution can increase the risk of developing PD.  These results are contained in Lisa Barnhill’s dissertation and the manuscript is in preparation.

An additional mechanism by which air pollution may increase the risk of PD is through increasing inflammation.  We did find that diesel exhaust did increase inflammation as measured by microglial activation.  We developed a computerized method to quantitate microglial activation in order to be able to compare the effects of diesel to other toxins. Interestingly, dopamine neurons were not protected when we reduced the number of microglia suggesting that inflammation is not important in dopamine neuron loss in our model at the times the fish were exposed.  

Cigarette smoking is another inhaled toxin but as opposed to air pollution, smoking is associated with a reduced risk of PD.  We tested cigarette smoke extracts in our zebrafish model and found that it too can kill dopamine neurons but there was almost no inflammation.  We are currently measuring cytokine levels in exposed fish to determine the inflammatory chemical response to toxins and their effect on autophagy.  These results support the theory that the inflammatory component of diesel exhaust increases the risk of PD.

Roles of Microglia in Diesel Exhaust Particulate Extract-Induced Neuron Loss and Clearance of α-Synuclein Aggregates in the Larval Zebrafish Brain (2018).  Murata, H., Barnhill, L., Khuansuwan, S, Park, A., Li, S. Bronstein, J.  March 12; Society of Toxicology, San Antonio TX.

Air Pollution and Risk of Parkinson’s Disease: Mechanistic Studies  (2018).  L. Barnhill, S. Khuansuwan, H. Murata, J. Araujo, JM. Bronstein.  Movement Disorders 32, Sept e9, Parkinson Study Group, Jersey City, NJ (rescheduled meeting due to hurricane).

A novel transgenic zebrafish line allows for in vivo quantification of autophagic activity in neurons (2018).  Sataree Khuansuwan, Lisa M. Barnhill, Sizhu Cheng, Jeff M. Bronstein.  Autophagy (in press).

September 2017 Project Update:

Epidemiological studies have implicated air pollution (AP) as a risk factor for developing PD and we are using various models to determine mechanisms by which it acts.  Extracts of diesel exhaust particles (DEPe) contain many of the suspected toxic components of AP and were used for all experiments.  

In human SH-SY5Y cells, DEPe exposure led to an increase in expression (mRNA) of α-synuclein, a central protein in the pathogenesis of PD.  In primary rat mesencephalic cultures, DEPe exposure led to an increase in α-synuclein protein levels in dopaminergic neurons.  Since increased levels of α-synuclein expression and protein levels have been clearly demonstrated to increase the risk of developing PD in humans, these data provide one potential mechanism for DEPe neurotoxicity.

Zebrafish (ZF) offer a unique opportunity to study mechanism of toxicity given that they are transparent and can be easily manipulated genetically.  We have found that DEPe exposure leads to dopamine neuron loss in ZF but unlike mammalian cells, it did not increase the expression (mRNA) of the ZF equivalent of α-synuclein, γ1-synuclein.  Despite this, we did find an increased level of γ1-syn aggregates in DEPe-treated ZF.  We hypothesized that these increased levels are due to decreased breakdown of the protein.  The ubiquitin proteasome degradation system was 1st tested but was unaffected by DEPe.  Using a newly developed transgenic ZF model (HuC-GFP-LC3) of the other degradation system (autophagy), we found marked alterations.  We are confirming these alterations using a newly developed assay of autophagic flux but if confirmed, these findings could explain the accumulation of γ1-syn and DEPe neurotoxicity in ZF.

Another important pathway implicated in PD is inflammation.  Using a transgenic ZF with labeled microglia (inflammatory cells in the brain), we found that DEPe markedly activates these cells.  Using genetic knockdown techniques and anti-inflammatory drugs, we are currently testing if microglia activation is responsible for at least some of DEPe toxicity to dopaminergic neurons.

In summary, we have found that DEPe exposure results alterations in a number of processes implicated in the pathogenesis of PD including increased levels of synuclein protein, disruption in protein degradation and induction in brain inflammation.  These studies add further support for the hypothesis that AP increases the risk of PD which could account for a large portion of PD cases.  Ongoing studies will determine which of these processes are essential mechanisms of DEPe toxicity and then we will target them with potential treatments.  These studies ultimately will provide important evidence that AP contributes to the development of PD so people can lower their exposure and provide legislators with additional evidence of the dangers of AP.  The mechanistic studies will guide us by providing targets for the development of treatments that slow or stop PD progression.

Air Pollution and Risk of Parkinson’s Disease: Mechanistic Studies  (2017).  L. Barnhill, S. Khuansuwan, H. Murata, J. Araujo, JM. Bronstein.  Movement Disorders (in press), Parkinson Study Group, Ft Myers Fl.

Extracts of diesel exhaust particles increase expression of α-synuclein.  L. Barnhill, S. Khuansuwan, J. Araujo, JM. Bronstein (in preparation).

Diesel exhaust extracts cause dopaminergic neuron damage by impairing autophagy. L. Barnhill, S. Khuansuwan, H. Murata, J. Araujo, JM. Bronstein (in preparation).

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