The goal of the project is to understand the dysregulation of inhibitory synapse caused by
poly-GR in C9ORF72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration
(FTD). Hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic
cause of both ALS and FTD. One potential pathogenic mechanism is the aberrant accumulation
of dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation in
all six reading frames (poly-GA, poly-GR, poly-PA, poly-PR and poly-PG) of both sense and
antisense RNAs. Lines of evidence indicate that some forms of the DPR proteins, particularly GR
and PR, can induce dysregulation of several molecular pathways, including stress granule
dysfunction, nucleocytoplasmic transport defects, altered ER homeostasis, et al. How these
molecular deficits influence neuronal functions remains understudied.
Accumulating evidence indicated that changes of synapse density and function occurs
prior to significant neuronal death in neurodegenerative diseases. The development of
hyperexcitability is a well-known phenomenon. One plausible mechanism is the alterations in the
ratio of excitatory to inhibitory activity (E/I imbalance). Our preliminary data identified specific
reduction of gephyrin clustering, a scaffold protein essential for inhibitory synapse formation and
function, in C9ORF72-ALS/FTD patient iPSC-neurons and in primary neurons with poly-GR
expression. We now propose to characterize the structure and electrophysiological dysfunction
of the inhibitory synapse, and to decipher the molecular mechanism how poly-GR causes such
defects by combining different –omics techniques with molecular and cellular approaches. We
aim to identify GR-interacting proteins in neurons using the proximity-labeling proteomics and
GR-induced transcriptomic changes by high-throughput sequencing, which will reveal candidate
genes/pathways for further mechanistic study of their contribution to GR-mediated synaptic
dysregulation. This project makes an important first step toward understanding the molecular
mechanisms of neuronal dysfunction in ALS and FTD patients. The molecular insights resulting
from this study will help understanding the etiology of the disease and developing novel
therapeutic targets.