A naturally occurring variant of SHLP2 is a protective factor in Parkinson's disease

Abstract

Mitochondrial DNA single nucleotide polymorphisms (mtSNPs) have been associated with a reduced risk of developing Parkinson's disease (PD), yet the underlying mechanisms remain elusive. In this study, we investigate the functional role of a PD-associated mtSNP that impacts the mitochondrial-derived peptide (MDP) Small Humanin-like Peptide 2 (SHLP2). We identify m.2158 T > C, a mtSNP associated with reduced PD risk, within the small open reading frame encoding SHLP2. This mtSNP results in an alternative form of SHLP2 (lysine 4 replaced with arginine; K4R). Using targeted mass spectrometry, we detect specific tryptic fragments of SHLP2 in neuronal cells and demonstrate its binding to mitochondrial complex 1. Notably, we observe that the K4R variant, associated with reduced PD risk, exhibits increased stability compared to WT SHLP2. Additionally, both WT and K4R SHLP2 show enhanced protection against mitochondrial dysfunction in in vitro experiments and confer protection against a PD-inducing toxin, a mitochondrial complex 1 inhibitor, in a mouse model. This study sheds light on the functional consequences of the m.2158 T > C mtSNP on SHLP2 and provides insights into the potential mechanisms by which this mtSNP may reduce the risk of PD.

Fig. 1. Identification of SHLP2 peptide in neuronal cells and demonstration of the importance of SHLP2 SNP in PD risk.

A Forest plot for meta-analysis (pooled random effects) of m.2158 T > C SNP on the prevalence of PD (B) A schematic diagram of m.2158 T > C SNP and SHLP2 gene in a long noncoding RNA of the mitochondrial DNA. C Microprotein-targeted mass spectrometry identified SHLP2 in SH-SY5Y cells. (Top) MS2 scan of endogenous SHLP2 peptide from C8 enriched SH-SY5Y lysate ran in PRM mode (Bottom) MS2 scan of the spiked-in heavy labeled SHLP2 peptide ran in PRM mode. R* represents non-radioactive heavy isotope labeling at Arginine residue. D Amino acid sequence of WT and K4R SHLP2 and the predicted 3D model of peptides obtained via the program PEP-FOLD3.

Fig. 2. SHLP2 localization and stability.

A Western blot of endogenous SHLP2 from subcellular fractions of HEK293 cells. B Extraction of endogenous SHLP2 from HEK293 mitochondria with buffer containing 1% Triton X-100 or Na₂CO₃ (C-E) HEK293 cells were transiently transfected with WT and K4R SHLP2 – tagged with EGFP. C mRNA expression of WT and K4R SHLP2 measured by qRT-PCR (D) (left) Representative image of total protein expression of WT and K4R SHLP2 (right) Quantification of the expression (E) Pulse-chase experiment of WT and K4R SHLP2. Cells were treated with 100ug/ml cycloheximide for the indicated time. p62/SQSTM1 is used as a loading control. Data are reported as mean ± SEM (n = 3/per group). Significant differences were determined by Student’s t-tests. *p < 0.05.

Fig. 3. SHLP2 is localized in mitochondrial complex 1 and co-expressed with mitochondrial genes.

A Mitochondrial localization of SHLP2-APEX. HEK293 cells were transfected with SHLP2-APEX or APEX control, fixed, and stained with anti-myc and anti-mitofilin antibodies to visualize APEX and mitochondria. Nuclei were stained with Hoechst. Scale bars are 200μm. B Representative western blot image of IMM and OMM proteins following SHLP2-APEX pulldown (n = 3). C Analysis of the SHLP2-APEX proximity labeling to remove false positives and perform pathway enrichment. D SHLP2 interacting proteins in the mitochondrial complex 1 (blue) SHLP2-APEX pull down (Red) SHLP2 peptide column. SHLP2 co-expression genes’ (E) molecular function and (F) cellular location in iPSC-derived dopaminergic neurons (iPSCs-derived dopaminergic neurons generated from 18 individuals). The color code represents the adjusted p value scale. The count is the number of genes significantly correlated for each term. The x axis is gene ratio, which is the ratio of input genes that are annotated in a term.

Fig. 4. K4R SHLP2, produced by individuals who carry the m. 2158 T > C polymorphism, has superior protection against mitochondrial dysfunction.

A TFAM expression and mtDNA copy number (B) NAD+ level was measured by mass spectrometry in WT and TFAM heterozygous knockout MEFs treated with 2.5μM WT and K4R SHLP2 for 24 hr. C (left) Immunostaining of mitochondria using COX IV antibodies. (right) The number of cells carrying mitochondria puncta was quantified. A total of 500-700 cells per group from four representative images were quantified. Scale bar is 50μm. D Cell viability was measured by MTT assay in SH-SY5Y cells treated with 2.5μM WT or K4R SHLP2 in the presence of 200μM MPP + . Data are reported as mean ± SEM (n = 4/per group). Significant differences were determined by Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = non significant.

Fig. 5. Pretreatment of mice with K4R SHLP2 shows protection in MPTP-lesioned mice.

A Schematic diagram of treatment. B Tyrosine hydroxylase (TH) expression in the striatum from mice treated with or without MPTP and WT SHLP2 and K4R variant. (n = 5 per group). C Schematic diagram of treatment. D Dopamine contents were measured by using high-performance liquid chromatography with electrochemical detection in the striatum from mice treated with or without MPTP and WT and K4R SHLP2 (n = 8 per group). Data are reported as mean ± SEM. Significant differences were determined by one-way ANOVA followed by Tukey’s post hoc test *p < 0.05 ** < 0.01**** < 0.0001.