TAR DNA-binding protein 43 (TDP-43) is of particular interest in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been speculated that loss of nuclear TDP-43 and its cytoplasmic aggregation contributes to neurodegeneration. Although considerable attention has been paid to RNA metabolism in TDP-43 function, TDP-43 is also known to act as a transcription factor. This study found that the expression of Nuclear-enriched abundant transcript 1 (NEAT1), a long-non-coding RNA, was substantially downregulated in motor neurons with nuclear TDP-43 loss, but upregulated in those with preserved nuclear TDP-43, in the postmortem spinal cords of patients with sporadic ALS. TDP-43 depletion induced Neat1 downregulation in Neuro2a cells, primary cortical neurons, and mouse spinal motor neurons. Furthermore, TDP-43 was found to positively regulate NEAT1 at the transcriptional level. Finally, Neat1 knockout exacerbates neurodegeneration of hSOD1(G93A) mice accompanied by increased misfolded superoxide dismutase 1 (SOD1) aggregations. Transcriptome analysis revealed that Neat1 knockout reduced protein folding-related genes, such as heat shock protein family A member 1A (Hspa1a), in the spinal cords of hSOD1(G93A) mice. Our results indicated that the loss of TDP-43 function enhances ALS neurodegeneration by losing the protective effect of NEAT1.