Investigating the Association Between TDP-43 Dependent Disruptions in RNA Metabolism and Neuronal Excitability

About This Grant

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by a progressive inability to control muscle movement. ALS patients are often comorbid with frontotemporal dementia (FTD), also known as ALS/FTD. The clinical manifestation of ALS/FTD is mediated by the selective dysfunction and degeneration of upper and lower motor neurons (MNs) that connect the CNS to the musculature, as well as cortical neurons that are responsible for speech and executive function. A broadly observed clinical feature in ALS/FTD patients is neuronal hyperexcitability. Motor neuron excitability, measured by transcranial magnetic stimulation and threshold tracking nerve conduction studies, is an early indicator of motor neuron disease that is detected pre-symptomatically, and is prognostic of disease progression and survival in both sporadic and familial cases. Despite its wide prevalence and therapeutic potential, the mechanisms that drive intrinsic neuronal hyperexcitability in ALS/FTD patients are poorly understood. Nuclear depletion and cytoplasmic aggregation of TDP-43 represents a unifying pathological feature of the overwhelming majority of ALS (~97%) and approximately 50% of all FTD patients (FTD-TDP). TDP-43 is a multifunctional RNA-binding protein that predominately resides in the nucleus and regulates RNA splicing. We have found that TDP-43 dysfunction causes spurious mis-splicing of KCNQ2 and UNC13A in ALS/FTD postmortem patient CNS tissue that strongly correlate with TDP-43 pathology, genetic predisposition, and clinical disease metrics. UNC13A encodes a protein with critical functions in synaptic vesicle release, while KCNQ2 encodes a potassium channel that regulates action potential frequency. Here, we will test the hypothesis that TDP-43 dependent splicing errors on UNC13A and KCNQ2 mRNAs trigger detrimental defects on distinct neuronal excitability properties that compromise their functionality and contribute to ALS/FTD pathophysiology. We will use induced pluripotent stem cell (iPSC)-derived cortical excitatory and inhibitory neurons, spinal motor neurons, and postmortem patient tissue to: 1) determine the extent and diversity of erroneous splicing of UNC13A and KCNQ2 in patients, 2) characterize the functional effects of these two events on neuronal physiology and, 3) develop single and bi- functional splice-modulating antisense oligonucleotides (ASOs) that can restore their normal splicing and neuronal physiology. In Aims 1 and 2 we will use single cell patch-clamp and population-based multi-electrode arrays to measure neuron intrinsic and network properties and determine the functional ramifications of TDP-43- mediated mis-splicing in human neurons. In Aim 3 we will design, screen, and identify splice-modulating ASOs conjugated by chemical linkers to simultaneously rescue both TDP-43 target mRNAs. Our studies will impact the field by highlighting a mechanistic link between TDP-43 dysfunction and neuronal excitability through aberrant mRNA metabolism, defining the functional ramifications of two of the most prevalent splicing errors of TDP-43, and by developing a new class of ASOs that could represent rational therapeutic modalities for ALS/FTD patients.