BLOC1S1 variants cause lysosomal and autophagic defects resulting in a hypomyelinating leukodystrophy with epileptic encephalopathy

Abstract

BLOC1S1 encodes a subunit shared by the BLOC-1 and BORC hetero-octameric complexes that regulate various endolysosomal processes. Here, we report the identification of seven distinct variants in BLOC1S1 in eleven individuals from seven independent families presenting with early psychomotor delay, hypotonia, spasticity, epileptic encephalopathy, optic atrophy, and leuko-axonopathy with hypomyelination. A subset of the affected individuals also have features of hypopigmentation and ocular albinism that are similar, although milder, than those of individuals with BLOC-1-related Hermansky-Pudlak syndrome. Functional analyses show that BLOC1S1 knockout (KO) impairs the anterograde transport of lysosomes and autophagy in both non-neuronal cells and iPSC-derived neurons. Rescue experiments reveal that most BLOC1S1 variants exhibit reduced expression, decreased assembly with other BORC/BLOC-1 subunits, and/or impaired restoration of lysosome transport and autophagy in BLOC1S1-KO cells. Additionally, we show that KO of BLOC1S1 reduces pigmentation in a melanocytic cell line, and that five of the BLOC1S1 variants partially or fully restore pigmentation. These findings provide genetic, clinical, and functional evidence that loss-of-function (LoF) of BLOC1S1 leads to more pronounced deficits in BORC than BLOC-1 function. We conclude that the biallelic BLOC1S1 variants characterized here primarily result in a neurological disorder with prominent leukodystrophy, similar to the recently reported condition caused by variants in the BORCS8 subunit of BORC. Together, these findings establish BORCopathies as a distinct disease entity.

Keywords: Autophagy; BLOC-1; BLOC1S1; BORC; Leukodystrophy; Lysosomes; Neurodevelopmental disorder.

Figure 1.. Clinical pedigrees and neuroimaging features of BLOC1S1-associated hypomyelinating leukodystrophy with epileptic encephalopathy

(A) Pedigrees showing segregation of variants (+) in the BLOC1S1 gene. In individual FI:1 there are two heterozygous variants in trans. In the remaining individuals (FII:1, FII:2, FIII:1, FIV:1, FV:1, FV:2, FVI:1, FVII:1, FVII:2a, FVII:2b) the variants are homozygous. (B) Brain MRI images corresponding to individuals FI:1, FII:1, FII:2, FIII:1, FV:1, FVI:1, and prenatal ultrasound images corresponding to Individuals FVII:1, FVII:2a, FVII:2b. MRI was not performed or imaging was not available for individuals FIV:1, FV:2, FVII:1. Brain MR images show variable hypomyelination with or without superimposed sequela of deep cerebral white matter dysmyelination or demyelination, corpus callosum abnormalities (hypoplasia, hypogenesis/dysgenesis, or agenesis), and brain volume loss. Fetal ultrsound images demonstrate ventriculomegaly and midline abnormalites. See Supplemental information for detailed image descriptions.

Figure 2.. Predicted structural features of BLOC-1 and BORC, and variant locations

(A) Amino-acid sequence of WT human BLOC1S1 (NP_001478.2) indicating the positions of the variants (red asterisks). (B) Amino-acid sequences of BLOC1S1 from 9 different species aligned to human BLOC1S1. Sequence conservation was calculated as a percent residue match between species with the human sequence, as described in the Materials and methods section, and represented graphically with a blue (low conservation between species) to maroon (high conservation between species) scale. (C) Structure of BLOC-1 predicted by AlphaFold Multimer. BLOC-1 subunits are shown in different colors. N- and C-termini of BLOC1S1 are indicated. (D) Close-up views of the positions of BLOC1S1 variants in BLOC-1 (highlighted in black). (E) Structure of BORC predicted by AlphaFold Multimer. BORC subunits are shown in different colors. N- and C-termini of BLOC1S1 are indicated. (F) Close-up views of the positions of BLOC1S1 variants in BORC (highlighted in black).

Figure 3.. Characteristics of BLOC1S1-KO HeLa cells

(A) SDS-PAGE and immunoblot analysis of WT and BLOC1S1-KO HeLa cells using antibodies to the endogenous proteins indicated on the right. α-tubulin was used as a control. Two isoforms of BLOC1S1 and the I and II forms of the autophagy protein LC3B are indicated by arrowheads on the right. The positions of molecular mass markers (in kDa) are indicated on the left. (B) WT and BLOC1S1-KO HeLa cells were fixed, permeabilized and immunostained for the endogenous lysosomal membrane protein LAMP1 and LC3B. Nuclei were labeled with DAPI (blue). Cell edges were outlined by staining of actin with fluorescent phalloidin or by background fluorescence (not shown, dashed lines). Images were obtained by confocal fluorescence microscopy. Scale bars: 20 μm.

Figure 4.. Characteristics of BLOC1S1-KO i3Neurons

(A) SDS-PAGE and immunoblot analysis of WT and BLOC1S1-KO i3Neurons using antibodies to the endogenous proteins indicated on the right. α-tubulin was used as a control. Only one isoform of BLOC1S1 is observed in these neurons. The I and II forms of the autophagy protein LC3B are indicted by arrowheads on the right. The positions of molecular mass markers (in kDa) are indicated on the left. (B, C) WT and BLOC1S1-KO i3Neurons were cultured for 20 days on glass coverslips, fixed, permeabilized and immunostained for the endogenous somatodendritic marker MAP2 (magenta), lysosomal membrane protein LAMP1 (grayscale), and mitochondrial protein TOMM2 (grayscale) (B), or MAP2 (magenta), the synaptic vesicle protein SYP1 (grayscale), and the dense-core vesicle protein CGA (grayscale) (C). Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The soma, dendrites and axons are indicated on the first image. (D) Top row: WT and BLOC1S1-KO i3Neurons were cultured for 20 days on glass coverslips, fixed, permeabilized and immunostained for endogenous MAP2 (magenta), LC3B (grayscale) and neurofilaments (NF) (grayscale). Nuclei were labeled with DAPI (blue). Scale bars: 20 μm. Bottom row: Enlarged images of the boxed areas in the upper row stained for the indicated markers. Scale bars: 20 μm. (E) Top row: WT and BLOC1S1-KO i3Neurons were cultured for 20 days on glass coverslips, fixed, permeabilized and immunostained with Tau-1 antibody (grayscale) and neurofilament heavy chain (NFH) antibody (grayscale). Scale bars: 20 μm. Bottom row: Enlarged images of the boxed areas in the upper row stained for the indicated markers. Scale bars: 20 μm. All images were obtained by confocal fluorescence microscopy.

Figure 5.. Assembly and lysosome-dispersal activity of BLOC1S1 variants

(A) HEK293T cells were transfected with plasmids encoding the indicated myc-tagged BLOC1S1 variants and subjected to immunoprecipitation with antibody to the myc epitope. Cell extracts (10%) and immunoprecipitates (myc IP) were analyzed by SDS-PAGE and immunoblotting (IB) for the myc epitope and the endogenous proteins indicated on the right. β-actin was used as a control. Arrowheads point to the specific proteins. The positions of molecular mass markers (in kDa) are indicated on the left. (B) Quantification from three to seven independent experiments such as that shown in panel A. Values are the mean ± SD. Statistical significance was calculated by one-way ANOVA followed by multiple comparisons using Dunnett’s test. *P < 0.05; **P < 0.01; ***P <0.001. (C) BLOC1S1-KO HeLa cells were co-transfected with plasmids encoding the indicated myc-tagged BLOC1S1 variants and GFP (to identify transfected cells; grayscale). Cells were subsequently fixed, permeabilized and immunostained for the endogenous lysosomal membrane protein LAMP1. Nuclei were labeled with DAPI (blue). Cell edges were outlined by staining of actin with fluorescent phalloidin (not shown, dashed lines). Images were obtained by confocal fluorescence microscopy. Scale bars: 20 μm. (D) Quantification by shell analysis of peripheral LAMP1 signal in BLOC1S1-KO HeLa cells expressing different BLOC1S1 variants from three experiments such as that shown in panel C. Data are represented as SuperPlots showing the individual data points, the mean from each experiment, and the mean ± SD of the means. Statistical significance was calculated by one-way ANOVA followed by multiple comparisons using Tukey’s test. ****P < 0.0001; ns: not significant.

Figure 6.. Autophagy-promoting activity of BLOC1S1 variants

(A) BLOC1S1-KO HeLa cells were co-transfected with plasmids encoding the indicated myc-tagged BLOC1S1 variants and GFP (to identify transfected cells; grayscale). Cells were subsequently fixed, permeabilized and immunostained for the endogenous autophagy protein LC3B. Nuclei were labeled with DAPI (blue). Cell edges were outlined based on background fluorescence (not shown, dashed lines). Images were obtained by confocal fluorescence microscopy. Scale bars: 20 μm. (B) Quantification of LC3B puncta per cell in BLOC1S1-KO HeLa cells expressing BLOC1S1 variants from three experiments such as that shown in panel A. Data were represented as SuperPlots showing the individual data points, the mean from each experiment, and the mean ± SD of the means. Statistical significance was calculated by one-way ANOVA followed by multiple comparisons using Tukey’s test. ***P < 0.001; ****P < 0.0001; ns: not significant.

Figure 7.. Pigmentation activity of BLOC1S1 variants

(A) SDS-PAGE and immunoblot analysis of WT and BLOC1S1-KO MNT-1 cells using antibodies to the endogenous proteins indicated on the right. α-tubulin was used as a control. Two variants of BLOC1S1 are indicated by arrowheads. The positions of molecular mass markers (in kDa) are indicated on the left. (B) WT and BLOC1S1-KO MNT-1 cell pellets. Notice the lighter pigmentation of the KO cells. (C) Flow cytometry of WT and BLOC1S1-KO MNT-1 cells. The x-axis indicates the number of cells normalized to the mode (or peak) of the graph. The y-axis indicates side scatter area (SSC-A). (D) Bar graph representing the geometric mean ± SD values of the SSC-A parameter from three independent flow cytometry experiments like that shown in panel C. (E) Bar graph representing the light absorbance at 405 nm measured after melanin extraction. BLOC1S1-KO MNT-1 cells were rescued with lentiviruses carrying the indicated protein. WT and BLOC1S1-KO MNT-1 cells were used as controls. Data from five or six independent experiments are represented as the mean ± SD. Statistical significance was calculated by one-way ANOVA followed by multiple comparisons using Tukey’s test. **P < 0.005; ****P < 0.0001; ns: not significant.