- Inhibitors of TDP-43 Aggregation and Toxicity in ALS
- Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS
Dr. Leonard Petrucelli's laboratory has pioneered neuroscience research aiming to understand the underlying mechanisms of amyotrophic lateral sclerosis (ALS) and identify potential drug targets for its treatment. TAR (Tat-responsive regulatory element) DNA-binding protein-43 (TDP-43) is a protein that has been found to go awry in approximately 90 percent of all ALS patients. Studies in yeast have revealed that C-terminal TDP-43 fragments are prone to aggregate and that only TDP-43 species that form inclusions, which result from continued protein aggregation, are toxic to neurons. With support from a Fiscal Year 2009 Therapeutic Development Award, Dr. Petrucelli is identifying compounds that prevent TDP-43 aggregation as potential neuroprotective agents for ALS. Using a previously developed human neuroblastoma cell line (M17D3) that overexpresses green fluorescent protein (GFP)-tagged C-terminal TDP-43 truncation product, GFP-TDP220-414, Dr. Petrucelli has begun screening compounds that reduce TDP220-414 aggregation, which is expressed as an attenuation of the GFP fluorescence. To increase the overall efficiency of the screening process, Dr. Petrucelli was able to effectively expand the assay from 24 to 384 wells. Over half of the 58,000 compounds from a select, proprietary small-molecule library have been screened on the M17D3 cells, and to date, 2,141 compounds have been found to attenuate GFP fluorescence (i.e., TDP fragment aggregation) by at least 30 percent. Expression of another relevant truncation product, GFP-TDP208-414, which can be exploited in M17D3 and also causes neurotoxicity in primary cortical neuronal cultures, is also being examined.
Future experiments will include further screening of the most promising compounds on primary cortical neuronal cultures. Expression of lactate dehydrogenase released into culture media will be measured as an indicator of cytotoxicity to further validate the compounds as potential therapeutic agents.
Amyotrophic lateral sclerosis (ALS) is a degenerative disease affecting the upper and lower motor neurons in the brainstem and spinal cord. Neural degeneration from ALS leads to progressive loss of voluntary muscle function, then to paralysis, and ultimately to death. A recent development in stem cell technology called induced pluripotent stem cells, iPSCs, is helping scientists understand the abnormalities in the cell biology behind ALS. iPSCs start as skin cells harvested from an ALS patient that are re-programmed in culture (through exposure to certain transcription factors), first into stem cells that have the capacity to become any type of cell, and then differentiated into glial-restricted precursor cells (iPSC-GRPs). These iPSC-GRPs act like neural developmental stem cells and can become motor neurons, astrocytes, or oligodendrocytes in culture. These cells may ultimately be transplanted into patients to treat ALS. Evidence exists suggesting that astrocytes and other non-neuronal cell types play a role in the neurodegeneration of ALS. Replacement of astrocytes derived from iPSC-GRPs may offer a technically and biologically more feasible treatment modality for ALS patients compared with motor neuron transplantation.
Using funding from a Fiscal Year 2009 ALS Research Program Therapeutic Development Award, Dr. Nicholas Maragakis of Johns Hopkins is initiating pre-clinical studies of iPSC-GRPs to assess their therapeutic potential. Dr. Maragakis will examine whether human iPSC-GRPs derived from either sporadic ALS (sALS), familial SOD1-mediated ALS (fALS), or normal control subjects have the same capacity for engraftment, survival, and neuroprotective qualities following transplantation. It is not known whether iPSC-GRPs from ALS patients will be normal (and thus possibly neuroprotective) or whether these cells may harbor ALS-specific abnormalities which may lack benefit, or possibly even exacerbate disease. By comparing normal iPSC-GRPs with sALS iPSC-GRPs and fALS iPSC-GRPs, Dr. Maragakis will attempt to reveal inherent differences in astrocyte biology related to ALS, providing potential insight into ALS disease mechanisms. This initial assessment of the therapeutic potential of these cells will help determine whether continued investigation of this concept is warranted. Being able to use a patient's own cells to treat ALS (autologous cell transplantation) could preclude the need for significant immunosuppression, as well as decrease the probability of cell rejection.
In another phase of the study, Dr. Maragakis will build on previous studies in rats where mutant SOD1-GRPs transplanted into the cervical spinal cord of normal rats demonstrated initial feasibility for the proposed methodology. In vivo studies in this project will examine the activity of the different types of iPSC-GRPs (sALS, fALS, and normal) following transplantation into the spinal cords of normal rats. Survival, differentiation, migration, and other properties will be examined across the different cell types. These same cells will also be transplanted into the SOD1G93A rat model of ALS, where they will be compared for survivability and effects on motor neuron survival and muscular function.
Dr. Maragakis' study lays the framework to answer initial questions about properties of iPSCs from ALS patients through in vitro and in vivo comparative studies. It also offers an initial assessment of potential neuroprotection in an SOD1 animal model of ALS. These studies could represent the initial development of a viable autologous cell therapy for ALS patients.