Arisla | The research

Active Project: TDP43-ASSEMBLY

TDP43-ASSEMBLY
Study of the aggregation process of TDP43 and analysis of the ability of the resulting aggregates to cause neuronal dysfunction

Amyotrophic lateral sclerosis (ALS) is manifest in two forms, sporadic (S-ALS) and familial (F-ALS), with 90% of individuals suffering from S-ALS. It is increasingly recognised that S-ALS is characterised by the conversion of the protein TDP-43 from its soluble state into insoluble aggregates that accumulate mainly in the cellular nucleus of motor neurons and in different areas of the central nervous system (brain and spinal cord).
A great deal of attention has been directed to the deposition of another protein, namely superoxide dismutase type I (SOD-1). Recent data indicate, however, that TDP-43 deposition is the major biochemical and hystopathological event in S-ALS, with SOD-1 involving mainly F-ALS. Only 261 papers have been published on TDP-43 with a focus on ALS, as opposed to the 2202 papers published on SOD-1 (Pubmed data).
The project described here is based on an integrated approach involving a biophysical investigation of the aggregation process of TDP-43 followed by a cell biological study of the ability of mature and intermediate protein aggregates to cause cell dysfunction.
We first propose to study the aggregation process of TDP-43 in real-time, using a variety of biophysical techniques that provide distinct, often complementary, morphological and structural information on the protein aggregates. These include far-UV circular dichroism, Fourier-Transform infrared spectroscopy, static and dynamic light scattering, electron microscopy, atomic force microscopy, thioflavin-T and Congo-red spectroscopic tests. This will allow an exploration of the intermediate and mature aggregates forming during the process and to investigate why TDP-43 form amorphous aggregates as opposed to the most favoured amyloid-like aggregates. Stopped-flow devices will also be used to detect early conformational changes occurring at the level of the monomeric protein.
The identified aggregates will be tested for their ability to cause cell dysfunction on both human neuroblastoma SH-SY5Y cells and primary rat neurons. Cell viability will be evaluated using the MTT reduction test, by measuring the level of ROS, the apoptotic status, etc.
The molecular and biological basis of neuronal dysfunction induced by TDP-43 aggregation will ultimately provide data useful to describe, at the molecular level, the pathogenic events of S-ALS. These findings would give useful information to develop new therapeutic strategies for S-ALS and related-disorders exhibiting TDP-43 pathology.

Research Team:

Principal Investigator:

Fabrizio Chiti