Accumulating evidence strongly suggest that amyotrophic lateral sclerosis (ALS) is linked to protein misfolding. TAR DNA–binding protein 43kDa (TDP–43) is nuclear RNA–binding protein, which is also implicated in the pathogenic cascades sporadic ALS by forming various aggregates. TDP–43 aggregates, as well as fused–in sarcoma (FUS) protein, are tightly linked to stress–granules, and the aberrant liquid phase separation attracts huge attention as a direct cause of aggregate toxicity. Therefore, the elimination of pathogenic forms of TDP–43 is a rational and a promising strategy to prevent motor neurons from degeneration. We previously generated a mouse monoclonal antibody (MAb), 3B12A raised against peptides containing E246/D247 located in the nucleus export signal (NES), which are exposed in the misfolded states of TDP–43. 3B12A MAb recognizes mislocalized and aggregated forms of TDP–43, but not non–aggregated WT TDP–43 in the nucleus. Using cDNA derived from mRNA of 3B12A hybridoma, we generated a single chain of variable fragments (scFv), aiming to eliminate intracellular pathogenic aggregates of TDP–43 in ALS. In the transfected cells, the 3B12A scFv showed the similar binding profiles to FL MAb, in which mislocalized or aggregated TDP–43, but not nuclear WT TDP–43 colocalized or co–immunoprecipitated with 3B12A MAb. Moreover, 3B12A scFv contains PEST–like motif at CDR2 of VH, which promotes the proteasome degradation of aberrant TDP–43, concomitant merit that the scFv alone was unstable in the absence of the antigen. To increase the degradation efficacy, we also tested the fusion of chaperone–mediated autophagy (CMA) signal to the 3B12A to provide dual proteolytic machinery. The 3B12A CMA–scFv acquired increased efficacy to shorten the half–life as well as to eliminate the TDP–43 aggregates in the transfected cells, estimated by the pulse–chase assay and time–lapse fluorescent microscope analysis, respectively. The scavenging of the intracellular aggregates by 3B12A CMA scFv also depressed the toxicity in HEK293A and Neuro2a cells. Notably, the interaction of CMA–scFv with the aggregates transcriptionally induced heat–shock chaperone, HSP70, which concomitantly refolded the misfolded TDP–43. In vivo evaluation of the neuroprotection of the intrabody, using the in utero electroporated mice pup brain, revealed that TDP–43 aggregates in the brain were significantly suppressed by the co–introduced intrabody, while expression levels of WT TDP–43 showed no significant change with or without the intrabody.
These results indicate that the misfolding–specific intrabodies with dual proteolytic signals promise molecular targeting therapy for TDP–43–linked ALS, although further estimation is required toward clinical application.
抄録全体を表示