Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration

Abstract

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.

Fig. 1. Mcl-1 deletion causes progressive white matter degeneration.

Brains of representative (A) Mcl-1^cKO or (B) Emx-Cre/Mcl-1^f/f mice shown in H&E-stained sagittal sections appear similar to controls at P7. By P14, Mcl-1^cKO mice develop white matter degeneration with ventricular enlargement (*) and abnormal migration of cerebellar granule neurons (arrows). White matter loss increases by P21.

Fig. 2. Mcl-1 deletion causes progressive neurologic impairment.

A Reduced weight gain in Mcl-1^cKO mice compared to controls. B Mcl-1^cKO mice show normal motor function at P7, with altered motor function by P14 and C increased hindlimb clasping at P18. D Mcl-1^cKO mice show increased startle in response to auditory stimuli at P14 and reduced elicited movement at P21. Amplitude was defined as the deflection produced by a piezoelectric transducer in the assay and integrates the startle movements into a quantifiable signal. Percent inhibition was calculated as 100 − [(response amplitude for prepulse stimulus and startle stimulus together/response amplitude for startle stimulus alone) × 100]. Time was defined as the delay between acoustic stimulus and startle responses. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001 respectively, relative to controls.

Fig. 3. Mcl-1 deletion causes white matter degeneration by increasing apoptosis.

A TUNEL demonstrates increased cell death in Mcl-1^cKO brains. For cell counts, one sagittal section from each replicate was scanned at ×20 and TUNEL+ cells were counted across the entire portion of each designated region that was contained within the section. All values are expressed as fold change in the number of TUNEL+ cells per standardized anatomic region, relative to the mean for the replicate control samples. B Representative H&E sections show that heterozygous Bak co-deletion and homozygous Bax co-deletion both rescue the white matter loss with ventricular enlargement (*) and cerebellar abnormalities caused by Mcl-1 deletion. Rescues were assessed relative to affected Mcl-1^cKO (Fig. 1A) and Mcl-1/Bax^fl/+ brains. C Quantification of BAK immunohistochemistry in corpus callosum shows that heterozygous Bak deletion significantly reduces the number of BAK+ cells. Quantification by detection of bioluminescence in western blot of whole cerebella shows that homozygous Bax deletion reduces BAX protein abundance. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively, relative to controls.

Fig. 4. Abnormal neuroimaging and myelination in Mcl-1-deleted mice, with different rescue effects caused by co-deletion of Bak or Bax.

A Representative MRIs show white matter hyperintensity in Mcl-1^cKO mice. B Total ventricular volume is abnormal in Mcl-1^cKO mice and increases from P7 to P21, while the volume of the fourth ventricle decreases significantly by P21. C, D Representative images and quantitative analysis of Myelin Basic Protein (MBP) IHC show that myelination is markedly decreased in Mcl-1^cKO mice, rescued in Mcl-1^cKO/Bak^+/− mice, and incompletely rescued in Mcl-1/Bax^dKO mice. D Graphs show MBP+ area/total area within individual sections, normalized to the mean value in the control mice. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively, relative to controls.

Fig. 5. Mcl-1 deletion causes loss of oligodendrocytes and Bergmann glia that is rescued with regional variation by co-deletion of Bak or Bax.

A, B Representative images and quantification of SOX10 and PDGFRA IHC in the indicated genotypes. C Representative immunofluorescence for GFAP and SOX2 IHC identify Bergmann glia in P14 mice of the indicated genotypes. D SOX2+ cells are significantly reduced in Mcl-1^cKO mice. * and ** denote p < 0.05 and p < 0.01, respectively, relative to controls.

Fig. 6. Mcl-1 deletion causes reactive astrocytosis.

A Representative GFAP IHC in sections of P14 cerebella in the indicated genotypes shows Bergmann glia processes in the control mice that are absent in the Mcl-1^cKO and numerous astrocyte processes in the Mcl-1^cKO mice. B Representative SOX9 IHC in sections of P14 corpus callosum in the indicated genotypes show a significant decrease of SOX9+ cells in the Mcl-1^cKO mice. C Quantification of SOX9 IHC at the indicated ages and genotypes, showing that astrocyte numbers are not increased in Mcl-1^cKO mice. D, E NESTIN/GFAP IHC, with representative images and quantification in the indicated genotypes. **denotes   p < 0.01 relative to controls.

Fig. 7. Progressive neuroinflammation in Mcl-1^cKO mice and reduced MCL-1 protein in mice with VWMD.

A, B IBA1 IHC, with representative images and quantification in the indicated genotypes, showing a progressive increase in microglia in Mcl-1^cKO mice, beginning in the corpus callosum at P14 and expanding to include diverse regions by P21. Co-deletion of Bak or Bax variably rescue microgliosis with different effects in different regions. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively, relative to controls. C Western blot comparing MCL-1 protein abundance in brain lysates of 7- and 10-month-old WT and eIF2B5^R132H/R132H mice, demonstrating reduced MCL-1 in 10-month-old mutant mice. * denotes p < 0.03, relative to controls.