Disruption of Golgi morphology and altered protein glycosylation in PLA2G6-associated neurodegeneration

Abstract

Background: Mutations in PLA2G6, which encodes the calcium-independent phospholipase A2 group VI, cause neurodegeneration and diffuse cortical Lewy body formation by a yet undefined mechanism. We assessed whether altered protein glycosylation due to abnormal Golgi morphology might be a factor in the pathology of this disease.

Methods: Three patients presented with PLA2G6-associated neurodegeneration (PLAN); two had infantile neuroaxonal dystrophy (INAD) and one had adult-onset dystonia-parkinsonism. We analysed protein N-linked and O-linked glycosylation in cerebrospinal fluid, plasma, urine, and cultured skin fibroblasts using high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization--time of flight/mass spectrometry (MALDI-TOF/MS). We also assessed sialylation and Golgi morphology in cultured fibroblasts by immunofluorescence and performed rescue experiments using a lentiviral vector.

Results: The patients with INAD had PLA2G6 mutations NM_003560.2: c.[950G>T];[426-1077dup] and c.[1799G>A];[2221C>T] and the patient with dystonia-parkinsonism had PLA2G6 mutations NM_003560.2: c.[609G>A];[2222G>A]. All three patients had altered Golgi morphology and abnormalities of protein O-linked glycosylation and sialylation in cultured fibroblasts that were rescued by lentiviral overexpression of wild type PLA2G6.

Conclusions: Our findings add altered Golgi morphology, O-linked glycosylation and sialylation defects to the phenotypical spectrum of PLAN; these pathways are essential for correct processing and distribution of proteins. Lewy body and Tau pathology, two neuropathological features of PLAN, could emerge from these defects. Therefore, Golgi morphology, O-linked glycosylation and sialylation may play a role in the pathogenesis of PLAN and perhaps other neurodegenerative disorders.

Keywords: Cell biology; Molecular genetics; Neuromuscular disease.

Figure 1

Mutation analysis of the biallelic PLA2G6 mutations in each patient. (A) Schematic overview of PLA2G6 transcripts encoding the two active isoforms, membrane bound iPLA2VIA-1 and cytosolic iPLA2VIA-2, and the mutations observed in the three patients. (B) gDNA sequence analysis of the region around the maternally inherited mutation NM_003560.2:c.950C>T in the PLA2G6 gene for each member of the family of patient 1. (C) Sequence analysis of the paternally inherited duplication of exons 4–7 (NM_003560.2:c.426–1077dup) with the resulting transcript and the location of the PCR primers on top. The cDNA amplification products, showing the exon 7–exon 4 junction in patient 1, are on the bottom. (D) gDNA sequence analysis of the region around the maternally inherited mutation NM_003560.2:c.2222G>A in the PLA2G6 gene for each member of the family of patient 2. (E) gDNA sequence analysis of the region around the paternally inherited synonymous mutation NM_003560.2: c.609G>A in the PLA2G6 gene for each member of the family of patient 2 and cDNA sequence analysis showing the splicing mutation encoding an in-frame 9 base pair deletion. (F) gDNA sequence analysis of the region around the maternally inherited mutation NM_003560.2:c.1799G>A in the PLA2G6 gene for each member of the family of patient 3. (G) gDNA sequence analysis of the region around the paternally inherited mutation NM_003560.2:c.2221C>T in the PLA2G6 gene for each member of the family of patient 3.

Figure 2

mRNA and protein expression analysis in fibroblasts. (A) Expression analysis of iPLA2GIVA-1 (dark bars) and iPLA2VIA-2 (light bars) mRNA levels in fibroblasts of each patient and a control cell line. Expression was normalised to GAPDH and plotted relative to the control. Error bars show SEM (B) Analysis of protein expression by western blot. GAPDH protein served as a loading control.

Figure 3

O-linked glycan profiles in cultured skin fibroblasts with and without overexpression of iPLAVIA-1. O-linked glycan profiles of control fibroblasts (A), control fibroblasts with iPLA2VIA-1 overexpression (B), fibroblasts from patient 1 (C), fibroblasts from patient 1 with iPLA2VIA-1 overexpression (D), fibroblasts from patient 2 (E), fibroblasts from patient 2 with iPLA2VIA-1 overexpression (F), fibroblasts from patient 3 (G) and fibroblasts from patient 3 with iPLA2VIA-1 overexpression (H). O-linked glycans are highlighted with dashed arrows. Samples were spiked with the internal standards (IS) C¹³-labelled T antigen and C¹³-labelled sialylated T.

Figure 4

Quantification of ER-Golgi intermediate compartment (ERGIC) and Golgi area by immunofluorescence and quantification of terminal sialic acid and terminal GlcNAc by lectin staining. (A) Graph of relative fluorescent intensity for Maackia amurensis lectin II (MAL II) and Sambucus nigra (elderberry) bark lectin (SNA) staining of terminal sialic acid. Data from the patient fibroblasts without (−) or with overexpression of iPLA2VIA-1 (1) or iPLA2VIA-2 (2) are plotted relative to that of control fibroblasts. Also see online supplementary figure S-2 and S-3. (B) Graph showing relative ERGIC-53 fluorescent intensity. Data from the patient fibroblasts without (−) or with overexpression of iPLA2VIA-1 (1) or iPLA2VIA-2 (2) are plotted relative to that of control fibroblasts. (C) Graph representing 2D-Golgi area of control fibroblasts and patient fibroblasts without (−) or with overexpression of iPLA2VIA-1 (1) or iPLA2VIA-2 (2). Golgi area was assessed by immunofluorescence staining with anti-GM-130 and anti-TGN-46. (D) Representative immunofluorescence images for ERGIC staining in Control and Patient 1 fibroblasts, with and without overexpression of iPLA2VIA-1 or iPLA2VIA-2. (E) Representative immunofluorescence images for staining of cis-Golgi with GM-130 and trans-Golgi with TGN-46 in Control and Patient 1 fibroblasts, with and without overexpression of iPLA2VIA-1 or iPLA2VIA-2. ^#p<0.05 versus control fibroblasts, *p<0.005 versus control fibroblasts, ⁺p<0.01 versus fibroblasts from the same patient without overexpression of PLA2G6, and ^†p<0.005 versus fibroblasts from the same patient without overexpression of PLA2G6. Error bars represent SEM.