2026 Theses Doctoral

An Unbiased Survey of the TDP43 Interactome in the Adult Brain Reveals its Scaffolding of Activity Dependent Synaptic Glycolysis

Wasserlein, Elizabeth Moran

TDP43 is an RNA-binding protein that regulates RNA processing. In multiple neurodegenerative diseases, TDP43 exhibits aberrant accumulation in the cytoplasm. TDP43 is often studied through the lens of its nuclear functions and considered ‘mislocalized’ in the cytoplasm. However, cytoplasmic TDP43 can also reflect regulated localization, enabling it to coordinate stimulus-responsive RNA and protein complexes across cellular compartments. Thus, the reductive reasoning that cytoplasmic TDP43 is mislocalized and that loss of nuclear function alone accounts for dysfunction, is oversimplified. It is unclear why TDP43 accumulation in the cytoplasm begins. Current theories include persistent stress driving cytoplasmic TDP43 into aggregates, but the ubiquitous expression of TDP43 makes it unclear why TDP43 dysfunction preferentially drives neurodegenerative disease. Together, these unresolved questions underscore how little is known about the physiological functions of TDP43. This is particularly true in the adult central nervous system, where understanding its normal functions is necessary to determine how TDP43 biology changes during neurodegenerative disease.

To address this, I adopted a proximity biotinylation approach, TurboID, to create TDP43Turbo and capture an unbiased TDP43-protein interaction network in the functioning adult brain. TDP43Turbo effectively identified well-established TDP43-associated RNA processing proteins, as well as novel synaptic protein interactions that enriched for proteins involved in other neurodegenerative disorders, most notably Alzheimer’s disease (AD). Compared to traditional approaches, TDP43Turbo detected proteins with larger disordered domains. This finding is significant because intrinsic disorder and the transient, dynamic interactions it enables are both central to TDP43 biology. I hypothesized that this technical improvement would allow TDP43Turbo to detect mutation-dependent changes to the TDP43 interactome, which have been inconsistently identified in the past, likely because traditional approaches were unable to capture these transient and dynamic TDP43 interactions. Using TDP43Turbo, I found that the amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) associated TDP43Q331K mutation interacted more with mitochondrial and synaptic proteins. The synaptic TDP43Q331K interactions alongside the novel TDP43 interactions associated with AD pointed to the possibility that synaptic TDP43 biology may be especially relevant to neurodegenerative disease.

To explore this further, I discovered that TDP43Turbo could capture protein interactions with a temporal resolution of 20 minutes, which allowed me to overcome difficulties in detecting both synaptic and activity-dependent TDP43 interactions. I found that TDP43 interactions at the synapse were primarily metabolic and that the metabolic interactions increased with activity. Markedly, the TDP43 interactions that responded most specifically to neuronal activity were glycolytic enzymes. Because glycolytic enzymes have been shown to assemble at synapses in response to neuronal activity, my preliminary mechanistic work showing that loss of TDP43 alters intraglycolytic organization suggested TDP43 may regulate the efficient local organization of glycolytic proteins at the synapse.

Orthogonal validation following voluntary running-wheel experiments showed that endogenous interactions between TDP43 and glycolytic proteins increase in a brain region-dependent manner and were often colocalized with cholinergic boutons, a synapse type known to facilitate memory and muscle contraction. Given the vulnerability of cholinergic synapses in ALS and AD, I also confirmed that interactions between TDP43 and glycolytic proteins are found in human postmortem cortical tissue. Together, this work supports a model in which TDP43 acts as an activity-dependent scaffold for glycolytic enzymes at cholinergic synapses and suggests that activity-dependent synaptic TDP43 function is highly relevant across neurodegenerative diseases.