Ongoing Projects
RNA based mechanisms in ALS and related neurodegenerative disease
Our studies have focused on TDP-43, an RNA binding protein involved in multiple aspects of RNA processing that associates with pathological inclusions in 97% of ALS and 45% of FTD cases regardless of etiology. Under normal conditions, TDP-43 is predominantly nuclear, however, in disease, it exits the nucleus and mislocalizes to the cytoplasm, consistent with both loss of nuclear function and gain of cytoplasmic function as possible toxicity mechanisms. We and others have shown that cytoplasmic TDP-43 associates with various RNA/protein complexes, including the translational repressor Fragile X Mental Retardation Protein (FMRP) (1), and it acts as a regulator of translation for specific mRNAs (2, 3 , 4). Our overarching goal is to define the role of TDP-43 in translation, identify its translational targets and determine their contribution to disease.A decade ago we developed Drosophila models of ALS based on overexpression (OE) of TDP-43 (WT or mutant) in motor neurons (MNs) or glia that recapitulate several aspects of the human disease including cytoplasmic inclusions, locomotor defects and reduced lifespan. Our approach is to take advantage of powerful molecular and genetic tools in the fly to uncover novel mechanisms of disease, then validate our findings in patient derived iPSC MNs, iPSC cortical neurons (iPSC CNs) and tissues. Using this strategy we identified alterations in the microtubule associated protein Futsch/MAP1B, the Hsc70-4/HSPA8 synaptic chaperone, the glypican Dlp/GPC6 and PFK, the rate limiting enzyme in glycolysis (2, 3 , 4, 5). These findings underscore the ability of our fly models to predict human disease relevant targets using a “fly-to-human” approach. Current efforts in the laboratory are aimed at establishing the functional relationship between TDP-43 and translation initiation factors using genetic interaction and molecular approaches such as NonCanonical Aminoacid Tagging (NCAT). Preliminary data show that enhancing translation mitigates ALS-like phenotypes in Drosophila models of TDP-43 proteinopathy including locomotor function and lifespan.
Modeling FTD in Drosophila
We recently developed a Drosophila model of FTD based on TDP-43 overexpression in mushroom bodies, a well characterized associative network in the fly brain, responsible for complex behaviors analogous to those controlled by cortical regions in humans.
Metabolic dysregulation in ALS
We have recently discovered that degenerating motor neurons upregulate glycolysis as a compensatory mechanism (5). Interestingly, it has recently become clear, that contrary to what was previously thought about metabolism in the CNS, neurons are capable of glycolysis and even assemble glycolytic enzymes at synapses to handle the demands of rapid synaptic communication or to handle stress. We found that phosphofructokinase (PFK), the rate limiting enzyme in glycolysis is significantly upregulated in a fly model of TDP-43 proteinopathy, patient derived iPSC motor neurons and spinal cords with TDP-43 pathology. Notably, over-expression of PFK is sufficient to rescue, while knocking down PFK aggravates TDP-43 dependent locomotor defects in motor neurons. Interestingly, it has been shown that PFK localizes to the neuromuscular junction of C. elegans under hypoxic stress. The clustering of PFK promotes synaptic vesicle recycling thereby maintaining synaptic function during times of high energy demand. Stressed yeast cells form glycolytic (G) bodies that contain PFK as well as chaperones, VCP and translation elongation/termination factors. Our findings about PFK in ALS and these recent reports about PFK suggest that in order to compensate for cellular energetics deficits caused by mitochondrial dysfunction, degenerating motor neurons reroute ATP production onto glycolysis to survive. We hypothesize that increased glycolysis may support the synaptic vesicle cycle and/or improve mitochondrial function via synaptic G bodies. To test this hypothesis we are working to: 1) Determine the mechanism by which PFK mitigates motor neuron dysfunction in ALS (i.e., improved synaptic vesicle cycle and/or mitochondrial function); 2) Determine G body composition and dynamics at synapses under normal conditions and in disease. Our “fly-to-human” approach enables us to first take advantage of powerful genetic tools and accessibility of the neuromuscular junction in flies then validate key findings in patient derived motor neurons. The results from these experiments will help formulate new hypotheses about neuronal metabolism and survival in disease, and the role of glycolysis in these processes.
Drug discovery using Drosophila models of human disease
Drosophila models of human disease provide low cost, rapid and effective strategies for in vivo drug screening. We have performed several drug screens to identify small molecules that mitigate TDP-43 dependent locomotor deficits, cytoplasmic aggregates and increase lifespan. We have sought to identify small molecules that target TDP-43 directly or unbiasedly mitigate TDP-43 dependent phenotypes. Ultimately, the goal of this project is to use phenotypic screening in vivo to identify ALS/FTD therapeutics.
Funding
National Institutes of Health
Department of Defense
Muscular Dystrophy Association
Private Donors