A Novel α-Synuclein K58N Missense Variant in a Patient with Parkinson's Disease

Abstract

Background: Parkinson's disease (PD) is a complex multifactorial disorder with a genetic component in about 15% of cases. Multiplications and point mutations in SNCA gene, encoding α-synuclein (aSyn), are linked to rare familial forms of PD.

Objective: Our goal was to assess the clinical presentation and the biological effects of a novel K58N aSyn mutation identified in a patient with PD.

Methods: We describe the clinical presentation associated with the novel mutation, together with genetic testing through whole exome sequencing (WES). Furthermore, we conducted extensive biophysical and cellular assays to assess the functional consequences of this novel variant.

Results: The patient exhibited typical features of sporadic PD with early onset and a benign disease course. WES showed a novel heterozygous missense variant in SNCA (NM_000345.4, c.174G>C; p.K58N). A positive family history of PD was evident, because both a parent and a grandparent had been diagnosed with PD but were deceased. The patient underwent deep brain stimulation surgery 13 years postdiagnosis, showing stable, long-term improvements in motor symptoms. Biophysical studies demonstrated K58N substitution causes local structural effects, disrupts membrane binding, and enhances aSyn in vitro aggregation. In cellular systems, K58N aSyn produces fewer inclusions per cell and does not form condensates. The variant increases aSyn cytoplasmic distribution and displays aberrant activity-dependent dynamic serine-129 phosphorylation.

Conclusions: The clinical presentation associated with the novel K58N aSyn mutation suggests a relatively benign PD course consistent with the phenotypic spectrum of idiopathic PD. Overall, our molecular studies provide novel insight into the biology and pathobiology of aSyn. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords: Parkinson's disease; genetics; neurodegeneration; protein aggregation; α‐synuclein.

FIG. 1

The p.K58N variant decreases the α‐helical content affecting liposome binding. (A) ¹H,¹⁵N Heteronuclear Single‐Quantum Coherence (HSQC) of α‐synuclein (αSyn) wild type (WT) (blue) and K58N (red). Evident Chemical shift perturbation (CSPs) are labeled. (B) Combined HN/N CSPs generated by the K58N mutation over the αSyn sequence. (C) Affected region is highlighted in gray. Hα (green) and Cα (black) CSPs. (D) Normalized ¹H,¹⁵N HSQC intensity ratios. Error bars are calculated from signal‐to‐noise ratios of individual resonances. (E) Secondary structure calculations from nuclear magnetic resonance (NMR) chemical shifts for αSyn WT (α‐helix, blue; β‐strand, light blue) and K58N (α‐helix, red; β‐strand, orange). On the right a zoom of the affected region with the α‐helical content is plotted. (F) Micelle‐bound αSyn structure (Protein Data Bank [PDB] ID: 1XQ8) with the positions of the different pathological mutations highlighted. (G) Residue‐specific α‐helical content over 100‐ns MD simulations. Error bars indicate the standard error from 40 analyzed peptides. (H–I) CD experiments of WT (H) and K58N (I) αSyn at different protein/lipid ratios. (J) Ellipticity change at 222 nm on increasing concentrations of lipids for WT (blue) and K58N (red). More negative values indicate a bigger increase of α‐helical structure. [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 2

K58N aSyn aggregation propensity. (A–D) In vitro ThT‐based aggregation assays for wild type (WT) and K58N α‐synuclein (aSyn). (A and B) Aggregation kinetic profiles of WT and K58N aSyn. Data were normalized to the maximum fluorescence value of each run. (C) Comparison of the half‐time in minutes for aggregation kinetics. (D) Comparison of maximum ThT fluorescence values of WT and K58N. Error bars represent mean ± SD. (E–G) Effect of K58N mutation on inclusion formation in cells. The patterns of inclusions formation were investigated using the SynT/Sph1 aggregation model, which is based on the coexpression of WT or K58N SynT variants and Sph1. (E) Immunohistochemistry images representing the inclusion formation in H4 cells expressing WT and K58N aSyn. Scale bars, 20 μm. (F) Quantification of the number of inclusions per cell and their area (G). For each experiment, 50 cells were counted at 100× original magnification. The data analysis was performed using a Student's t test and presented as mean ± SD (N = 3). *P < 0.05, **P < 0.01. [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 3

Characteristics of wild‐type (WT) and K58N α‐synuclein (aSyn) filaments. (A) Transmission electron microscopy (TEM) micrograph of WT aSyn amyloid filaments. Black arrows mark selected filament ends. Scale bar, 100 nm. (B) TEM micrograph of K58N aSyn amyloid filaments. Black arrows indicate filament ends of exemplary filaments. Scale bar, 100 nm. (C) Two‐dimensional (2D) class averages (706‐Å box size) of twisting WT aSyn filaments, illustrating the interaction between either two protofilaments (2PF) or a single protofilament (1PF). (D) 2D class averages (706‐Å box size) show twisting K58N aSyn filaments, again characterized by two protofilaments interacting with each other. Scale bar, 50 nm. (E) Overview of the electron density map of WT aSyn filaments. (F) Overview of the electron density map of K58N aSyn filaments. (G) Amino acid sequence of human aSyn with distinct regions color‐coded (N terminus in orange, nonamyloid component in purple, and C terminus in green). (H) The electron density map together with the atomic model of WT aSyn amyloid filaments featuring a single beta‐sheet layer formed by two interacting protofilaments. The protofilament interface is stabilized by a K45‐E57 salt bridge. (I) The electron density map together with the atomic model of K58N aSyn amyloid filaments. The protofilament interface is again stabilized by the K45‐E57 salt bridge. The mutated residue is indicated in red. (J) Superposition of the WT atomic model (gray) and the K58N mutant (blue). Although the double‐arrow structure is similar, notable differences in the backbone highlight the impact of the K58N mutation (residue marked in red) on the overall aSyn filament structure. [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 4

aSyn K58N shows decreased condensate formation in vitro and in cells. (A) aSyn phase separation in the presence of 2 mM Ca²⁺ and crowding with 15% PEG‐8000, immediately after PEG addition for aSyn wild‐type (WT) and the disease variant aSyn K58N. aSyn concentration used: 100 μM. (B) Heatmap for turbidity measurements of aSyn phase separation in the presence of 2 mM Ca²⁺. Data are derived from four independent repeats. (C) Condensate formation of aSyn WT YFP and aSyn K58N YFP on ectopic expression with VAMP2 in HeLa cells. aSyn K58N YFP does not undergo condensate formation in cells. (D) Quantification of condensate formation. Data were derived from incuCyte screening, 16 images per well, three wells per biological repeat, three biological repeats. n indicates biological repeats. Data are represented as mean ± SD. Unpaired two‐tailed t test. [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 5

Dynamic activity‐dependent phosphorylation at serine 129 (pS129) of K58N and wild‐type (WT) α‐synuclein (aSyn). (A) Schematic illustration of aSyn structure exhibiting the KTKEGV repeat sequence harboring most familial PD mutations, central hydrophobic domain, and C‐terminal region. The sequence alignment of aSyn is displayed below showing the conserved KTKEGV residues in yellow and sites of familial PD mutation in orange. On the right, the experimental setup is summarized. (B) A representative Western blot (WB) displaying the levels of total aSyn and pS129 from DIV 17–21 rat SNCA ^−/− cortical neurons expressing WT and K58N aSyn, with WB quantitative analysis presented below. (C) WB results of WT and K58N transduced rat SNCA ^−/− cortical neurons that were subjected to on‐plate sequential extraction to isolate the cytosolic (C) and membrane (M) fractions. MJFR1 antibody was used for total aSyn detection, whereas GAPDH and Calnexin were used as cytosolic and membrane fractions controls, respectively. (D) Quantitative analysis of WT and K58N aSyn solubility from WB data in (C). (E) A summary of the experimental conditions to study pS129 dynamic reversibility, with more information provided in the main text. (F and G) Neuronal activity‐dependent reversible phosphorylation of S129 (as outlined in E) was detected in DIV 17–21 rat SNCA ^−/− cortical neurons expressing WT or K58N, respectively, after picrotoxin (PTX) stimulation (20 μM) and tetrodotoxin (TTX) inhibition (1 μM). WB for quantifying total aSyn and pS129 was employed, and (H)–(L) are derived from (F) and (G). (H) The percentage of increase in pS129, compared with baseline, for WT and K58N aSyn observed after 2 or 4 h of PTX stimulation. (I) Comparison of TTX‐resistant pS129 levels in WT and K58N variants relative to baseline conditions (DMSO vehicle). (J) The proportion of irreversible pS129 relative to the basal (DMSO vehicle) condition. (K) The proportion of irreversible pS129 relative to 2 h of PTX stimulation. (L) The proportion of irreversible pS129 relative to 4 h of PTX stimulation. ****P < 0.0001, ***P < 0.001, *P < 0.05. The error bar was mean ± SD. ns, not significant. [Color figure can be viewed at wileyonlinelibrary.com]