Astrocytes are central in the pathomechanisms of vanishing white matter

Abstract

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders.

Figure 1. Generation of VWM mouse models.

(A) 2b5^ho mice were generated by introducing a construct into the Eif2b5 gene locus consisting of exons 4–6, including a point mutation in exon 4. (B) 2b4^ho mice were generated by introducing a construct into the Eif2b4 gene locus consisting of exons 12 and 13, including a point mutation in exon 13. (C) Kaplan-Meier graph shows the reduced lifespan of VWM mutant mice, with an average survival of 19 months for 2b4^ho mice (n = 9); 8 months for 2b5^ho mice (n = 19); 4 months for 2b42b5^he/ho mice (n = 12); and 3 weeks for 2b4^ho2b5^ho mice (n = 6). (D) Staining for MBP showed vacuolization of the cerebellar white matter in 7-month-old 2b5^ho, 4-month-old 2b42b5^he/ho, and P21 2b4^ho2b5^ho mice, but not in 7-month-old WT mice. Scale bars: 50 μm. (E) The distribution of axonal diameters was skewed to the smaller diameters in 2b5^ho and 2b42b5^he/ho mice (n = 92 WT mice; n = 100 2b5^ho mice; n = 91 2b42b5^he/ho mice). Immunostainings are representative images from at least 3 experiments.

Figure 2. White matter astrocytes are immature and have abnormal morphology and intermediate filament composition.

(A) Nestin-positive cells were present in the corpus callosum of 7-month-old 2b5^ho mice (middle), but not in that of 7-month-old WT mice (left). Double staining for GFAP confirmed that these cells were astrocytes (right). (B) The number of nestin-positive cells in the corpus callosum of 2b5^ho mice increased from P14 onward as the disease progressed (n = 30, WT mice; n = 21, 2b5^ho mice). (C) In all VWM mutant mice, the number of nestin-positive cells in the corpus callosum was significantly increased (n = 16, 19-month-old 2b4^ho mice; n = 6, 4-month-old 2b42b5^he/ho mice; n = 3, P21 2b42b5^ho mice). (D) GFAPδ protein levels were increased in forebrain lysates from 2b5^ho mice at all ages examined. (E) Staining for GFAPδ showed increased immunoreactivity in white matter astrocytes from mutant mice compared with those from WT mice. (A and E) Scale bars: 50 μm. (B and C) *P < 0.05 and **P < 0.01 by Mann-Whitney U test. Each data point in B indicates 1 mouse with a trend line; data points in C represent the ratio of mutant over WT, with the solid data point indicating the mean ratio of mutant over WT ± SEM. Immunostainings are representative of at least 3 experiments.

Figure 3. VWM astrocytes inhibit OPC maturation in vitro.

(A) Compared with cocultures of WT astrocytes and WT OPCs (left), cocultures of 2b4^ho astrocytes and WT OPCs (middle) showed a decrease in MBP- and MOG-positive cells. Cocultures of 2b5^ho astrocytes and WT OPCs showed the lowest number of MBP- and MOG-positive cells (right). (B) Lower numbers of MBP- and MOG-positive cells were present in cocultures with 2b4^ho astrocytes, independent of the OPC genotype (WT or 2b4^ho). No differences were seen between cultures with WT astrocytes and WT or 2b4^ho OPCs. Scale bars: 50 μm (A and B). (C) The number of MBP-positive cells was significantly decreased in cocultures with 2b4^ho astrocytes (n = 8). There were no significant differences between cultures with WT or 2b4^ho OPCs (n = 6). (D) A similar pattern was observed for MOG-positive cells. (C and D) Data points indicate 1 experiment, with the solid data point indicating the mean ± SEM. *P < 0.05, by paired-samples t test. Immunostainings are representative images of at least 3 experiments.

Figure 4. WT ACM rescues OPC maturation.

(A) Immunostaining for olig2, MBP, GFAP, and MOG showed decreased immunoreactivity of MBP and MOG in the cocultures with 2b4^ho ACM, but not with WT ACM. Scale bars: 50 μm. (B and C) The numbers of MBP- (B) or MOG-positive (C) cells were significantly lower in cocultures with WT and 2b4^ho astrocytes when grown in 2b4^ho ACM, but the numbers increased with exposure to WT ACM (n = 4 for all). (B and C) “wk refresh” indicates refreshment of the medium once per week, as was done for all other cocultures; “d refresh” indicates a daily refreshment of the medium, as a control for the daily refreshment in the conditioned medium experiments. (D) The amount of hyaluronan was significantly increased in 7-month-old 2b5^ho mice, but not in 1-month-old or 4-month-old 2b5^ho animals (n = 5 for all). (E) ACM of WT and 2b4^ho mice showed no significant differences in hyaluronan levels, although in two 2b4^ho mutant mouse ACM samples, the hyaluronan levels were greatly increased. After treatment with hyaluronidase, no hyaluronan signal was detected by ELISA in any sample (n = 6 for all). (B, C, and E) Data points indicate 1 experiment, with solid data points indicating the mean ± SEM. (D) Data points represent the ratio of mutant over WT, with solid data points indicating the mean ratio of mutant over WT ± SEM. *P < 0.05 and **P < 0.01, by paired-samples t test (B and C), Student’s t test (D), and Mann-Whitney U test (E). Immunostainings are representative images of at least 3 experiments.

Figure 5. Astrocyte pathology outside the brain white matter.

(A) In the cerebellum of 7-month-old 2b5^ho and P21 2b42b5^ho/ho mice, Bergmann glia overexpressed GFAPδ, with displacement of the cell bodies to the molecular layer and abnormal morphology with short, thick cell processes. (B and C) A similar Bergmann glial pathology was also present in VWM patients as shown in double stainings for GFAP and GFAPδ (B) and S100β (C). (D) Histology of WT retina showed linear organization of the retinal layers. a, ganglion cell; b, inner plexiform; c, inner nuclear; d, outer plexiform; e, outer nuclear; f, photoreceptor. VWM mutant retinae showed increasing laminar disorganization, with ectopic neurons in the plexiform layers and severe displacement of outer nuclear cells in the P21 2b4^ho2b5^ho animals (bottom). (E) Staining against GFAP showed decreased immunoreactivity in the outer plexiform layer of retinae from 2b5^ho mice, whereas the P21 2b4^ho2b5^ho mouse retinae contained Müller cells with coarse processes spanning across the outer nuclear and photoreceptor layer. (F) Staining against glutamine synthetase (GS) showed markedly reduced immunoreactivity in the Müller glia of the 2b5^ho and 2b4^ho2b5^ho mutants. Scale bars: 50 μm (A, C, E, and F); 20 μm (B); 200 μm (D). Immunostains are representative images of at least 3 experiments.