Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease with no known etiology. The formation of pathological protein inclusions, including RNA-binding proteins such as TDP-43 and rho guanine nucleotide exchange factor (RGNEF) are a hallmark of ALS. Despite intensive research, the mechanisms behind protein aggregate formation in ALS remains unclear. We have investigated the role of metabolic stress in protein aggregate formation analyzing how it is relevant to the co-aggregation observed between RGNEF and TDP-43 in motor neurons of ALS patients. Metabolic stress was able to induce formation of micronuclei, small nuclear fragments, in cultured cells. Notably, we observed the formation TDP-43 protein inclusions within micronuclei that co-aggregate with RGNEF and can be released to the cytoplasm. We observed that the leucine-rich domain of RGNEF is critical for its interaction with TDP-43 and localization in micronuclei. Finally, we described that micronuclei-like structures can be found in brain and spinal cord of ALS patients. This work is the first description of protein inclusion formation within micronuclei which also is linked with a neurodegenerative disease. The formation of TDP-43 inclusions within micronuclei induced by metabolic stress is a novel mechanism of protein aggregate formation which may have broad relevance for ALS and other neurodegenerative diseases.