Usage of an Alternative Translation Start Site in PRKN mutation carriers: Key to Later-Onset Parkinson’s Disease and a Novel Therapeutic Target

Biallelic pathogenic variants of PRKN, encoding the Parkin RBR E3 ubiquitin protein ligase, are the most common known cause of autosomal recessive Parkinson’s disease (PD). PARK- PRKN is characterized by an early median age at onset (AAO) of 31 years with a wide range (3-81 years). When evaluating the 16 previously published carriers of a homozygous deletion of Exon 2 ( PRKN ^delEx2 ) from the MDSGene database, the median AAO is later (39.5 years; range: 25-75 years) than in carriers of other PRKN pathogenic variants. Here, we investigated 26 homozygous PRKN ^delEx2 patients, including 20 from additional sources, and confirmed the later median AAO (37 years, range: 13-60 years). Furthermore, we investigated one carrier who was still unaffected in her 60s. To elucidate the functional basis for this observation, we used induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (iDN) from this unaffected carrier as well as genome-edited iDNs from two control lines and neuroblastoma cell lines (SH-SY5Y) with the introduction of a homozygous PRKN ^delEx2 . We observed elevated levels of an N-terminally truncated form of Parkin (Parkin ^Δ1–79) , starting at an internal translation initiation site (TIS) in Exon 3 (p.Met80 in the full-length protein) in cells with the Exon 2 deletion. Furthermore, in silico prediction and Parkin quantification in isogenic neuroblastoma cell lines suggested the presence of residual Parkin ^Δ1–79 also for other PRKN variants upstream of the alternative TIS. iDNs from PRKN ^delEx2 carriers partially retained Parkin E3 ubiquitin ligase activity in contrast to carriers of other exonic deletions in PRKN ( PRKN ^delEx3 , PRKN ^delEx7 ) via the expression of Parkin ^Δ1–79 . Importantly, endogenous Parkin E3 ubiquitin ligase activity of Parkin ^Δ1–79 was enhanced in neurons derived from a homozygous PRKN ^delEx2 variant carrier upon treatment with a small molecule allosteric modulator of Parkin (BIO-2007817). In summary, the later AAO in patients with homozygous PRKN ^delEx2 is associated with partially retained Parkin function. The residual ligase activity can be further increased pharmacologically, providing mutation-specific personalized counseling opportunities and a potential novel therapy for selected patients with PARK- PRKN .