Grants & Funding > American Parkinson Disease Research Grants 2016

American Parkinson Disease Association is using its 2016 distribution to fund:

Pharmacological activation of TFEB using a small molecule promotes autophagic clearance of aggregated alpha-synuclein: Implications for Parkinson’s disease

Identifying pathogenic translational targets of G2019S in dopamine neurons

 

PROJECT TITLE:  Pharmacological activation of TFEB using a small molecule promotes autophagic clearance of aggregated alpha-synuclein: Implications for Parkinson’s disease.

Investigator/Author:  Shankar J. Chinta, PhD

Objective:  To test the therapeutic potential of our recently discovered novel TFEB-inducing compounds using well established in vitro and in vivo mouse models of a-syn oligomerization

Background:  Lysosomal dysfunction has been associated with neuropathology not only in rare lysosomal storage diseases (LSDs), but also in more common age-related neurodegenerative disorders including PD. In the case of PD, defective lysosome function has been shown to result in impaired clearance of α-syn, resulting in its accumulation in the form of soluble oligomers and aggregates. Recent studies from our laboratory and others have demonstrated that function of the master regulator of the autophagy-lysosomal pathway (ALP), the transcription factor EB (TFEB), is significantly reduced in PD patients as well as in animal models displaying α-syn accumulation. This suggests that defects in the autophagy-mediated clearance of α-syn contribute to the progressive loss of SNpc neurons characteristic of the disorder. As the master regulator of lysosomal biogenesis, TFEB plays a crucial role in autophagy and the maintenance of intracellular protein homeostasis. Recent studies by Decressac et al. demonstrated that TFEB is co-localized with alpha-synuclein oligomers in affected postmortem tissues from PD patients. Further in vivo studies suggested that alpha-synuclein may elicit its pathological effects by ‘trapping’ TFEB in the cytoplasm, leading to impaired transcription of lysosomal genes, inhibition of ALP function and, hence, decreased alpha-synuclein clearance. They further demonstrated that reducing TFEB levels exacerbated alpha-synuclein toxicity whereas TFEB overexpression was able to restore ALP function, enhance clearance of α-syn oligomers, and reduce neurodegeneration. Collectively, these findings suggest that impairment of TFEB is a key element in the pathogenesis of PD, making TFEB a very promising therapeutic target for counteracting autophagy dysfunction and neurodegeneration in PD and related disorders.

Methods/Design:    In order to test the therapeutic efficiency of these newly identified potent TFEB inducers, we propose to initially test their ability to rescue dopamine neurons from α-syn toxicity in PD patient iPSC-derived neurons. This is based on the rationale that these cells are functionally closer to cell types found in the diseased human PD brain. We will investigate the ability of the TFEB inducers to prevent alpha-synuclein oligomerization, increased oxidative stress, and dopaminergic neuronal death. Finally, we will investigate whether autophagy is involved in TFEB-mediated reduction of α-syn aggregates in these cells by monitoring a series of autophagic markers upon treatment with these novel compounds.

In our second set of experiments, we will assess the efficiency of these compounds in vivo synuclein overexpression model. For these studies, we will use the well-established wildtype α-syn AAV-mediated rat model, which exhibits several cardinal features of PD neuropathology. Following 3 weeks of compound treatment, we will assess whether these TFEB inducers prevent the accumulation of (1) oligomeric α-syn inclusions in TH+ neurons, (2) TH+ striatal fiber density and stereological SNpc cell counts, and (3) behavioral analyses (cylinder test, amphetamine-induced rotation). These studies will provide us with evidence as to the efficacy of these agents to stimulate autophagy as a viable therapeutic approach for PD that merits further research to move forward to the clinical stage.


Relevance to Diagnosis/Treatment of Parkinson’s Disease:  Recent discovered in Dr. Chinta’s laboratory and others demonstrated that function of the master regulator of the autophagy-lysosomal pathway (ALP), the transcription factor EB (TFEB), is significantly reduced in people with PD as well as in animal models displaying alpha-synuclein accumulation.  This study will test the therapeutic potential of TFEB-inducing compounds using well established in vitro and in vivo mouse models of alpha-synuclein oligomerization

This research study aims to provide evidence that pharmacological activation of TFEB reduces the accumulation of aggregated alpha-synuclein and promotes alpha-synuclein clearance by enhancing the autophagy pathway. This study will hopefully provide proof-of-principle evidence that pharmacological activation of TFEB is a viable therapeutic strategy to enhance the degradation of alpha-synuclein aggregates and may hold great promise for disease intervention in PD and potentially other neurodegenerative diseases characterized by protein deposition.

September 2017 Project Update:

We recently performed a screen for compounds that can boost the cells ability to get rid of misfolded proteins via lysosomal degradation.

During the past year, we initially tested the therapeutic potential of recently discovered compounds using in vitro (in the test tube) cell model derived from Parkinsonian patients. Initial studies with these cell lines have shown that compound significantly boost cells ability to degrade the misfolded alpha synculein aggregates. The compound treatment also showed significant induction of lysosomal   regulator transcription factor EB (TFEB) (cell functioning).

We next examined the ability of this compound in well-established mouse model of Parkinson’s disease. These mice overexpress Parkin mutation (Q311X) specifically in dopaminergic neurons and they exhibit Parkinsonian phenotype when they reach 16 months of age. Parkin Q311X mutant mice and their WT (wild type) littermates were fed either normal chow or chow containing compound C1 starting at 13 months of age for a 12 week period. As expected, Parkin Q311X mutant mice used in our studies by 16 months of age, displayed several parkinsonian neuropathologies, including DAergic cell loss, accumulation of insoluble (proteinase K-resistant) α-synuclein, and motor deficits. Following chronic C1 treatment, there is a significant protection of dopaminergic neurons in Parkin mice. C1 treatment also significantly reduced alpha synuclein aggregates and improved behavioral deficits in these mice. In the coming year, we are in the process of evaluating the markers of cell destruction in samples from these mice.

Back to Top

PROJECT TITLE:  Identifying pathogenic translational targets of G2019S in dopamine neurons

Investigators/Authors:  Ian Martin, PhD

Objectives: The overall objective of the project is to assess how wild type and mutant forms of LRRK2 associated with PD affect translational profiles of different neuronal sub-populations using transgenic Drosophila melanogaster models. Candidate translational targets of LRRK2 will be assessed for their role in pathogenic LRRK2 phenotypes associated with PD.

Background: Considering the high energetic cost of protein production to cell, regulated protein translation plays an essential role in supporting organismal development and maintenance under variable conditions. Accumulating evidence supports a multifaceted and complex role for LRRK2 in regulating protein homeostasis by directly impacting autophagic protein turnover, vesicular protein trafficking and the biosynthesis of proteins via ribosomal protein phosphorylation. The common PD-linked G2019S mutation in LRRK2 has been shown to cause an increase in bulk protein translation via hyper-phosphorylation of ribosomal protein s15 in Drosophila brain.  Blocking this through protein synthesis inhibitor treatment or phosphor-deficient T136A s15 expression prevents the age-related loss of dopamine neurons and locomotor deficits observed in G2019S LRRK2 transgenic flies. Translational profiling of mutant LRRK2 flies may uncover targets that are important in neurodegeneration.

Methods/Design: Translating ribosome affinity purification (TRAP) will be used to perform genome-wide translational profiling in the whole nervous system and specifically in dopamine neurons and other neuronal subpopulations of aged flies. Genetic and pharmacologic manipulation of candidate translational targets will be used to assess their role in mutant LRRK2 toxicity.

Relevance to Diagnosis/Treatment of PD: The objectives of this research are to 1) study the effects of altered dietary amino acids on dopamine neuron degeneration and locomotor impairment associated with mutant G2019S LRRK2, and 2) to assess the role of TOR signaling in the effects of dietary amino acids on G2019S LRRK2-induced phenotype.

The study will examine the role of diet-influenced TOR activity and protein translation on dopamine neuron viability in aging Drosophila. If metabolically-influenced protein translation is found to contribute to dopamine neuron death in aging organisms, this will be informative in understanding potential mechanisms of PD development in humans. Additionally, if restricting dietary amino acids can alleviate the effects of G2019S LRRK2 on dopamine neuron loss and motor dysfunction, this will open the window to manipulation of dietary amino acids, which has been well studied in fields of aging and cancer, as a possible way to manage LRRK2-linked PD.

September 2017 Project Update:

We study mutant forms of the gene LRRK2 that have been shown to cause Parkinson’s disease. We are interested in identifying how mutant forms of this gene impact the process of protein translation (production) in the dopamine neuron cells in the brain. We used a specialized technique obtaining cell specific gene profiles (translating ribosome affinity purification TRAP) to measure the effects of mutant LRRK2 expression on the protein translation profiles of those neurons in our model organism – Drosophila melanogaster (fruit fly). From the data obtained, we identified several genes whose translation appears to be increased by mutant LRRK2 that we are pursuing as candidates to underlie mutant LRRK2 toxicity in dopamine neurons. We are currently validating the findings of our translational profiling (identifying molecular changes of a gene in response to alterations) experiments using additional independent methods (quantitative real-time PCR) then will proceed to assessing their role in neurodegenerative phenotypes (specific characteristics) observed in mutant LRRK2 expressing flies.

Back to Top

Click for a printer friendly version

Back