Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity

Apolipoprotein (apo) E4, a 299-aa protein and a major risk factor for Alzheimer's disease, can be cleaved to generate C-terminal-truncated fragments that cause neurotoxicity in vitro and neuro-degeneration and behavioral deficits in transgenic mice. To investigate this neurotoxicity, we expressed apoE4 with C- or N-terminal truncations or mutations in transfected Neuro-2a cells. ApoE4 (1-272) was neurotoxic, but full-length apoE4(1-299) and apoE4(1240) were not, suggesting that the lipid-binding region (amino acids 241-272) mediates the neurotoxicity and that amino acids 273-299 are protective. A quadruple mutation in the lipid-binding region (1250A, F257A, W264R, and V269A) abolished the neurotoxicity of apoE4(1-272), and single mutations in the region of amino acids 273-299 (L279Q, K282A, or Q284A) made full-length apoE4 neurotoxic. Immunofluorescence staining showed that apoE4(1272) formed filamentous inclusions containing phosphorylated tau in some cells and interacted with mitochondria in others, leading to mitochondrial dysfunction as determined by MitoTracker staining and flow cytometry. ApoE4(241-272) did not cause mitochondrial dysfunction or neurotoxicity, suggesting that the lipid-binding region alone is insufficient for neurotoxicity. Truncation of N-terminal sequences (amino acids 1-170) containing the receptor-binding region (amino acids 135-150) and triple mutations within that region (R142A, K146A, and R147A) abolished the mitochondrial interaction and neurotoxicity of apoE4(1-272). Further analysis showed that the receptor-binding region is required for escape from the secretory pathway and that the lipid-binding region mediates mitochondrial interaction. Thus, the lipid- and receptor-binding regions in apoE4 fragments act together to cause mitochondrial dysfunction and neurotoxicity, which may be important in Alzheimer's disease pathogenesis.