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[Preprint]. 2024 Jun 17:2024.06.17.599406.
doi: 10.1101/2024.06.17.599406.

The Role of Alpha-Synuclein in Synucleinopathy: Impact on Lipid Regulation at Mitochondria-ER Membranes

Peter A Barbuti  1   2   3   4 , Cristina Guardia-Laguarta  1   2 , Taekyung Yun  1   5 , Zena K Chatila  1 , Xena Flowers  6   7 , Bruno Fr Santos  3   4   8 , Simone B Larsen  3 , Nobutaka Hattori  9 , Elizabeth Bradshaw  1   6   7 , Ulf Dettmer  10 , Saranna Fanning  10 , Manon Vilas  1   6   11 , Hasini Reddy  6   12 , Andrew F Teich  1   6   12 , Rejko Krüger  3   4 , Estela Area-Gomez  1   2   5   6 , Serge Przedborski  1   2   12   13
Affiliations

The Role of Alpha-Synuclein in Synucleinopathy: Impact on Lipid Regulation at Mitochondria-ER Membranes

Peter A Barbuti et al. bioRxiv. .

Abstract

The protein alpha-synuclein (αSyn) plays a critical role in the pathogenesis of synucleinopathy, which includes Parkinson's disease and multiple system atrophy, and mounting evidence suggests that lipid dyshomeostasis is a critical phenotype in these neurodegenerative conditions. Previously, we identified that αSyn localizes to mitochondria-associated endoplasmic reticulum membranes (MAMs), temporary functional domains containing proteins that regulate lipid metabolism, including the de novo synthesis of phosphatidylserine. In the present study, we have analyzed the lipid composition of postmortem human samples, focusing on the substantia nigra pars compacta of Parkinson's disease and controls, as well as three less affected brain regions of Parkinson's donors. To further assess synucleinopathy-related lipidome alterations, similar analyses were performed on the striatum of multiple system atrophy cases. Our data show region-and disease-specific changes in the levels of lipid species. Specifically, our data revealed alterations in the levels of specific phosphatidylserine species in brain areas most affected in Parkinson's disease. Some of these alterations, albeit to a lesser degree, are also observed multiples system atrophy. Using induced pluripotent stem cell-derived neurons, we show that αSyn contributes to regulating phosphatidylserine metabolism at MAM domains, and that αSyn dosage parallels the perturbation in phosphatidylserine levels. Our results support the notion that αSyn pathophysiology is linked to the dysregulation of lipid homeostasis, which may contribute to the vulnerability of specific brain regions in synucleinopathy. These findings have significant therapeutic implications.

Keywords: Alpha-synuclein; Parkinson’s disease; dopaminergic neuron; lipid metabolism; mitochondria-associated ER membranes; neurodegeneration.

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Conflict of interest statement

Competing Financial Interests SP is a member of the scientific board of Luciole Pharmaceuticals and a reviewing editor for eLife.

Figures

Figure 1:
Figure 1:. Lipid alterations in the postmortem Parkinson’s disease (PD).
(A) Heatmaps representing 2-fold changes in the abundance of individual lipid species of (A) esterified cholesterol (CE) (B) ceramide (Cer), (C) phosphatidylcholine (PC) and (D) phosphatidylserine (PS) across different brain regions: substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), hypothalamus (Hypo), substantia innominata (SI) of PD postmortem donors compared to controls.
Figure 2:
Figure 2:. Lipid alterations in patient-derived neurons expressing differing alpha-synuclein (αSyn) doses.
(A) Lipidomic heat maps showing Log2 fold changes in lipid groups and selected individual lipid species in induced pluripotent stem cell (iPSC)-derived neurons carrying either αSyn duplication (αSyn-Dupl) or αSyn knock out (αSyn-KO), normalized to age-matched neurons expressing endogenous αSyn levels (Ctrls) after 30 days of directed differentiation. Relative concentrations of total sphingolipids (bottom left) and sphingolipids containing long-chain fatty acids (22:1 to 26:1) (bottom right) are presented, normalized to the concentrations in Ctrl samples. Data are presented as the mean ± SEM of at least 3 independent biological replicates (n) analyzed by an ordinary two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Total sphingolipids: Interaction (F16,99 = 5.462; ****p<0.0001) and Row Factor (cell line) (F2,99 = 1.743; p=0.1804). Sphingolipids containing long-chain fatty acids: Interaction (F16,99 = 1.522; p=0.1068) and Row Factor (cell line) (F2,99 = 19.09; ****p<0.0001). (B, top) Lipidomic heat maps displaying Log2 fold changes in selected phospholipids. (Bottom) Relative concentrations of total phospholipids and phospholipids containing polyunsaturated fatty acids (PUFAs, 40:4 to 42:7) are presented, normalized to the concentrations in Ctrl samples. Data are presented as the mean ± SEM of at least 3 independent biological replicates (n) analyzed by an ordinary two-way ANOVA, followed by Tukey’s multiple comparison test. Total phospholipids: Interaction (F18,110 = 2.485; **p=0.002) and Row Factor (cell line) (F2,110 = 1.684; p=0.1904). Sphingolipids containing long-chain fatty acids: Interaction (F18,110 = 5.215; ****p<0.0001) and Row Factor (cell line) (F2,110 = 67.56; ****p<0.0001).
Figure 3:
Figure 3:. Lipid alterations in neurons with alpha-synuclein (αSyn) duplication across subcellular fractions
(A) Qualitative (left) and quantitative (right) assessments of monomeric αSyn distributions in the total unfractionated homogenate and in mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), ER, and cytosol fractions from induced pluripotent stem cell (iPSC)-derived neurons carrying different αSyn levels after 30 days of directed differentiation. A semi-quantitative assessment of αSyn abundance was performed using protein immunoblots. Protein levels were normalized to the levels of long-chain fatty acid–CoA ligase 4 (Acsl4, MAM marker), calnexin (bulk ER marker), or protein kinase C (PKC, cytosolic marker). Data are presented as the mean ± SD of at least 3 independent biological replicates (n) analyzed by an ordinary one-way analysis of variance (ANOVA). MAM (Interaction F3,10 =32.56 ****p<0.0001); ER (Interaction F3,9 =17.21 ***p=0.0005); Cytosol (Interaction F3,10 =9.424 **p=0.0029). (B) Lipidomic heat maps displaying Log2 fold changes in groups and individual lipid species of sphingolipids; neutral lipids, and phospholipids in αSyn-Duplication iPSC-derived neurons compared with controls (Ctrls) in the total unfractionated homogenate and in MAM and ER fractions. In Ctrl neurons, alterations in sphingolipid species were consistent between MAM and ER fractions (right). (C) MAM and ER fractions show elevated phosphatidylcholine and phosphatidylserine concentrations, but a specific reduction in phosphatidylserine lipid species comprised of smaller-chained hydrocarbons is also observed. (D) Individual neutral lipids species across the total homogenate, MAM and ER fractions highlights notable differences in diglyceride (DG) and triglyceride (TG) lipids between fractions.
Figure 4:
Figure 4:. Different alpha-synuclein (αSyn) levels alter mitochondria-associated endoplasmic reticulum membrane (MAM) function.
(A) Phospholipid synthesis in induced pluripotent stem cell (iPSC)-derived neurons. Quantification of de novo phosphatidylserine (PS) synthesis and the ratio phosphatidylethanolamine (PE) to PS (PE/PS) levels in patient-derived neurons after 30 days of directed differentiation after incubation with 3H-Serine (3H-Ser) for the indicated times. Data were normalized to the Control cell line, and at least 4 independent differentiations were performed. KO SNCA, SNCA knock out. A repeated measures two-way analysis of variance (ANOVA) with assumed sphericity was performed for the indicated experiments: PS synthesis: Column (Cell line) factor (F2,10 =6.777; *p=0.0138); Row (time) factor (F2,20 = 10.92; ***p=0.0006); PE/PS: Cell line factor (F2,11 = 13.41; **p=0.0011); time factor (F2,22 = 1.180; p=0.3260). (B) Protein levels of PS synthase (PSS)1 and PSS2 at the MAM domain. Analysis of MAM fractions from iPSC-derived neurons probed for PSS1 and PSS2, with a representative image shown, adapted from (81). Data are presented as the mean ± SD of at least 4 independent biological replicates (n) analyzed by an ordinary one-way ANOVA of the indicated proteins: PSS1 (Interaction F3,22 =1.326; p=0.2912); PSS2: (Interaction F3,22 =10.46; ***p=0.0002). Tukey’s multiple comparison was used for post hoc analysis with a single pooled variance. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PC, phosphatidylcholine. (C) Assessment of PSS2 activity. Quantification of de novo PS synthesis from 14C-PE in patient-derived neurons after 30 days of directed differentiation. After subcellular fractionation and quantification, 100 μg of protein isolated from the crude mitochondria was incubated with 14C-PE for 45 minutes. Lipids were immediately extracted using the chloroform/methanol extraction method followed by a modified Bligh and Dyer protocol. A minimum of 4 independent differentiations were performed, and data were normalized to the Control cell line. A repeated measure one-way ANOVA with assumed sphericity was performed. Column (Cell line) factor (SS: 0.4430; DF: 2; MS: 0.2215; F2,6 =12.23; **p=0.0076).
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