The normally nuclear protein TDP-43 accumulates in the cytoplasm of neurons in many people with amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND) and frontotemporal dementia (FTD). However, the drivers of TDP-43 dysfunction and the biological factors and pathways that modify these processes remain unclear. We have therefore developed and studied a diverse set of cell, human neuron, and mouse models of TDP-43 dysfunction. These include viral systems for expression of human wildtype and disease-mimicking TDP-43 variants, CRISPR/Cas9 approaches for fluorescent tagging of endogenous TDP-43, and analysis of transgenic TDP-43 mice. We have shown that disease-mimicking TDP-43 variants form highly mobile intranuclear structures or stable cytoplasmic inclusions that sequester endogenous TDP-43, suggesting that pathological conversion of TDP-43 drives dysfunction of normal TDP-43. Quantitative proteomics and CRISPR screening approaches have identified hundreds of genes and proteins that differentially associate with, or regulate formation of, pathological TDP-43. Further, we have identified TDP-43-pathology associated alterations in diverse pathways including protein degradation, apoptotic signalling, and cell stress pathways throughout disease. Together, these studies reveal involvement of distinct and overlapping biochemical pathways in formation of TDP-43 pathology and associated neurotoxicity, which may guide development of targeted therapeutics for ALS and FTD.