Jim Himelic Foundation: Eradicate ALS Jim Himelic Foundation: Eradicate ALS

Please encourage friends to join JHF in its fight against ALS. Any size donation would be extremely appreciated.Please encourage friends to join JHF in its fight against ALS. Any size donation would be extremely appreciated.

Research

We research the various steps in RNA processing including transport and translation during normal development and aging of neurons, as well as during the onset and progression of disease. In addition to these basic studies we seek to identify therapeutic strategies for diseases linked to RNA and metabolic dysregulation in the nervous system. Our research utilizes a combination of genetic, molecular, bioinformatics and pharmacological approaches in Drosophila (fruit flies), cultured cells and patient tissues. This “fly-to-man” approach takes advantage of the powerful, genetically tractable fruit fly model to uncover molecular mechanisms that we can subsequently validate in patient tissues.

 

(Left) Using simple genetic models to develop treatments for human disease, by CC, UA Art Department

Projects

RNA Processing in amyotrophic lateral sclerosis Metabolic Dysregulation in ALS Age-dependent alterations in RNA stress granules and cognitionDrug discovery using Drosophila models of human disease

RNA Processing in ALS

 

Several RNA binding proteins have been found in proteinaceous aggregates, a hallmark of neurodegeneration, which suggests that they are involved in the pathomechanism of disease. Additionally, some have been shown to harbor mutations causing amyotrophic lateral sclerosis (ALS) and/or related neurodegenerative diseases such as fronto-temporal degeneration (FTD).  Among these, TDP-43 is linked to the vast majority of ALS cases (>95%) and has been implicated in RNA splicing, transport, storage in RNA stress granules (SGs) and translation. These data suggest intimate links between TDP-43, RNA metabolism and disease. Our overarching hypothesis is that TDP-43 acts as a regulator of mRNA localization and translation in motor neurons, and that dysregulation of these processes contributes to the pathophysiology of ALS. This hypothesis is tested using a combination of molecular, genetic, imaging, bioinformatics and functional approaches. We expect this research to provide novel insights into TDP-43’s function in translation, to identify physiologically significant and disease relevant protein partners and mRNA targets, which in turn may pinpoint much needed molecular targets and pathways with therapeutic potential for ALS and related neurodegenerative diseases.

RNA binding protein partners of TDP-43Using a Drosophila model of ALS we developed we recently demonstrated that Fragile X Protein (FMRP), an established translational regulator that our group has previously worked on, is neuroprotective by reducing TDP-43 aggregation and restoring the translation of specific mRNA targets (Figure 1).

Figure 1. A model for TDP-43 – FMRP interaction. Similar to what has been observed in the human disease, TDP-43 overexpression (OE) results in formation of RNA SGs that persist. These RNA SGs cause sequestration of mRNA targets and translation inhibition. FMRP OE is neuroprotective and modulates TDP-43 solubility. FMRP OE restores TDP-43 dependent translation inhibition of specific mRNA targets.

Our current efforts are aimed at understanding the physical interactions between TDP-43, FMRP and other RNA binding proteins in temporary mRNA-storage stress granules (SGs), including granule components and translation initiation factors that we have identified using biochemical and/or genetic approaches. These experiments are expected to provide a mechanistic view of TDP-43’s role in translation and identify strategies for remodeling RNA granules with small molecules (see Drug Discovery in Drosophila paragraph below).

RNA targets of TDP-43

In addition to searching for protein partners, we have sought to identify mRNA targets of TDP-43 in neurons.  We recently showed that TDP-43 regulates the localization and translation of futsch/MAP1B mRNA in Drosophila. The defects we identified in the ALS model fly motor neurons are remarkably similar to those found in human ALS spinal cord neurons (Figure 2). This is significant because on one hand, dysregulation of Futsch/MAP1B offers an explanation for the microtubule and synaptic instability found in ALS and on the other, it provides proof of principle that our “fly-to-man” approach can be effective in elucidating the pathomechanism of neurodegeneration.

Figure 2. Microtubule-stabilizing protein Futsch/MAP1B is retained in motor neuron cell bodies. Top panels: motor neurons showing increased protein in ALS flies (top right) compared to controls (top left). White arrowheads indicate motor neuron cell bodies. Bottom panels: spinal cords from ALS patients (bottom right) show higher levels of Futsch/MAP1B compare to controls (bottom left). Black arrowheads indicate motor neuron cell bodies.

 To identify neuronal specific mRNA targets and to determine what stage of translation is impacted by TDP-43 expression, we are using RNA immunoprecipitations (RIP) and translating ribosome affinity purification (TRAP). Using RIP and TRAP, we have identified several novel candidate translational targets including hsc70-4 mRNA, which encodes a molecular chaperone that controls synaptic vesicle (SV) trafficking, as well as additional candidates implicated in synaptic function. Notably, restoring Hsc70-4 levels in motor neurons by overexpression rescues synaptic vesicle endocytosis defects caused by ALS-associated mutant TDP-43 (TDPG298S).

Figure 3. A model for TDP-43 and Hsc70-4 interactions. (A) In controls, TDP-43 does not sequester mRNA targets or protein partners leading to normal levels of mRNA translation and synaptic proteins at the NMJ. As a result, SVC occurs as normal. (B) Motor neuron expression of TDP-43 results in decreased synaptic expression of Hsc70-4 and defects in SV endocytosis. Mutant TDP-43 sequesters hsc70-4 mRNA in insoluble complexes and inhibits its translation. Note: post-transcriptional reduction in Hsc70 expression and SVC deficits are also present in C9orf72 models of ALS.

These findings suggest that ALS is caused at least in part by dysregulation of translation of key mRNAs regulating the synaptic vesicle cycle at the neuromuscular junction, consistent with ALS being “a synaptopathy”. The identification of hsc70-4 mRNA, a key cellular chaperone as a translational target of disease-associated mutant TDP-43 protein and a component of TDP-43 aggregates, is exciting because it unifies the ribostasis (i.e., RNA processing) and proteostasis (i.e., protein folding) hypotheses of neurodegeneration.

We are currently investigating additional mRNA candidate targets encoding among others synaptic and metabolic proteins.

Metabolic dysregulation in ALS

Nourishing the brain, by Brittany Squires, UA Art Department

Long-standing observations made in the clinic point to defects in metabolic regulation including abnormalities in glucose and lipid metabolism in ALS. Using our fruit fly model of ALS based on TDP-43 we have performed global metabolomics profiling and identified several significant metabolic changes consistent with alterations in cellular energetics. Specifically, increased pyruvate in both TDPWT and disease-associated TDPG298S models is suggestive of altered glucose metabolism. We also found increased tricarboxylic (TCA) cycle intermediates that just like pyruvate, are upregulated in plasma from ALS patients. Additionally, increased levels of fatty acid carnitine conjugates suggest decreased lipid beta-oxidation. Consistent with this observation, the ketone body marker 3-hydroxybutyrate (BHBA) is decreased, implying impaired mitochondrial lipid metabolism and an increased reliance on glycolysis for energy production in affected motor neurons. Based on these results and our previous published findings that antidiabetic drugs show partial effectiveness in ALS (see Drug discovery in Drosophila) we hypothesize that improving glucose and lipid metabolism through diet and genetic intervention can provide protection against neurodegeneration.

We have evidence that various dietary and genetic interventions aimed at increasing ATP production mitigate TDP-43 dependent locomotor dysfunction and increase lifespan. Transcriptional profiling data for key glycolytic enzymes are consistent with increased glycolysis in both fly and patient-derived iPS motor neurons. Together with genetic interaction experiments, data collected to date support the notion that increased glycolysis is a compensatory mechanism, perhaps a last resort for motor neurons to counter the well documented reduction in cellular ATP/dysfunctional mitochondrial metabolism caused by TDP-43 toxicity. Interestingly, these exciting findings tie in with recent reports that glycolytic enzymes cluster at synapses and, under stress conditions, regulate synaptic vesicle endocytosis, a process we found altered also through our studies on hsc70-4 mRNA translation. To determine whether increased reliance on glycolysis is specific to TDP-43 or represents a common mechanism, we are testing whether a high glucose diet improves locomotor function and increases lifespan in additional models of ALS, based on SOD1 and C9ORF72. So far we are finding that at least locomotor function is also improved in these other models by high glucose availability. We are continuing our “fly-to-man” approach to determine the contribution of glucose and lipid metabolism to ALS phenotypes and to establish the feasibility of pursuing therapeutic strategies aimed at restoring energy homeostasis.

Age-dependent alterations in RNA stress granules and cognition

On the hunt for genes and small molecules that help neurons live longer and healthier, by Elena Makansi UA Art Department

Aging is the biggest risk factor for neurodegenerative disease and is garnering attention due to increased life expectancy. The ability of aging bodies to handle stress is diminished considerably by specific effects on multiple cellular processes including decreased translation. We have preliminary observations that RNA binding proteins associated with RNA stress granules and translation are altered in aging fly brains. In collaboration with Dr. Carol Barnes at University of Arizona we are beginning to explore correlations between RNA stress granules, aging and cognition in flies and rodents.

Drug discovery using Drosophila models of human disease

Small molecule screens

One of the principal reasons for the high costs of drug development is the failure in the toxicity and/or efficacy stages when a drug is tested in vivo. Fly models can be used for screening entire libraries of compounds, for validating lead therapeutics quickly and effectively, and for testing drug toxicity and efficacy in vivo for various human diseases. We have developed a screen for neuroprotective compounds in our ALS models (Patent Application No. 14/508,610, pending) and screened both 1,200 FDA approved compounds.  Among these we found that pioglitazone, a drug approved by the FDA to treat diabetes mellitus can improve locomotor function but does not extend lifespan in ALS flies. A similar result was reported in clinical trials, which showed that pioglitazone has no significant improvement in survival despite promising results in a SOD1 mouse model of ALS. In retrospect, the results in the fly model were similar to the results of the human trials, suggesting that perhaps fly models could provide a better, more cost-effective and rapid prediction of outcome in clinical trials. In addition, we are testing “new chemical matter” compounds synthesized by our collaborator, Dr. Jon Njardarson (Chemistry and Biochemistry, University of Arizona) and identified four small molecules with neuroprotective potential in vivo. We are currently validating these small molecules in the fly model and plan to test them in a mammalian model in the near future. We are also collaborating with biotech companies to screen compounds of interest.

Rational design

In collaboration with Drs. May Khanna (Pharmacology) and Vijay Gokhale (Bio5) we are using a combination of in silico docking, biophysical studies and in vivo validation assays in Drosophila to synthesize small molecules that restore the expression of TDP-43 target mRNAs.

Hunt for small molecules, by Michael Divine, UA Art Department

   

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