Figure 6.

NENF Association with DJ-1 and PINK1 in Neurogenesis

(A) Model of NENF role with DJ-1 and PINK1 in neurotrophic activity and neuronal survival/death; question mark indicates the mechanistic pathway in which NENF secreted is unclear.

(B) PINK1 and DJ-1 immunoprecipitates in whole-cell extract (WCE) as well as in mt, ER, and MAM lysates from ECSCs, DNLCs, and mouse brain using anti-PINK1 and DJ-1 antibodies immunoblotted (IB) with anti-NENF antibody (i). mt, ER, and MAM markers (ii) used as internal control.

(C) Representative confocal micrographs of NENF co-localization (arrow heads) with DJ-1 and PINK1 in the rat SN region using antibodies specific to these native proteins and mt marker (AIF, apoptosis-inducing factor); DNA visualized by DAPI (blue), scale bar, 20 μm.

(D) NENF secretion in the conditioned medium and cell lysates immunoblotted (IB) with anti-NENF antibody.

(E) Secreted NENF measured in control (ctrl) single-guide RNA (sgRNA) and single KOs of ECSCs or DNLCs.

(F) Disruption of NENF association with DJ-1 and PINK1 in the indicated fractions of KOs versus control sgRNA in DNLCs (i) or in the fibroblasts of patients with PD with PINK1 mutation versus healthy control (ii). Band intensities normalized to loading control (LC).

(G) Heme measured in DNLCs of control sgRNA and indicated KOs with NENF immunoprecipitates.

(H) Neurotrophic (cell viability, neurite outgrowth) and apoptotic activities (BCL-2, caspase-9) in control versus KOs, CRISPR-resistant (CR) genes fused with FLAG in indicated KOs versus control, or DJ-1 wild-type (WT) versus catalytic (E163K) mutant fused with FLAG in DJ-1 KO DNLCs. Data in (E, G, and H) are mean ± SD (n = 3 biological replicates; *control versus single KOs or DJ-1 WT versus variant (E163K) with p ≤ 0.05 by Student's t test).

(I) DJ-1 (PDB ID: 3SF8) with potential interface residues (bold) binding with NENF. Immunoprecipitates of DJ-1 WT or catalytic mutants fused with FLAG in DJ-1 KO DNLCs using anti-FLAG antibody IB with anti-NENF or FLAG antibody. Band intensities normalized to LC.

See also Figure S6.