The unfolded protein response modulates disease severity in Pelizaeus-Merzbacher disease

Abstract

The unfolded protein response (UPR) is a eukaryotic signaling pathway linking protein flux through the endoplasmic reticulum to transcription and translational repression. Herein, we demonstrate UPR activation in the leukodystrophy Pelizaeus-Merzbacher disease (PMD) as well as in three mouse models of this disease and transfected fibroblasts expressing mutant protein. The CHOP protein, widely known as a proapoptotic transcription factor, modulates pathogenesis in the mouse models of PMD; however, this protein exhibits antiapoptotic activity. Together, these data show that the UPR has the potential to modulate disease severity in many cells expressing mutant secretory pathway proteins. Thus, PMD represents the first member of a novel class of disparate degenerative diseases for which UPR activation and signaling is the common pathogenic mechanism.

Figure 1. The UPR Is Induced in Cells Expressing Mutant PLP1

(A) Transfected COS-7 cells expressing mutant (Aa) but not wild-type (Ab) PLP1 (green) localize CHOP (red) in the nucleus (blue). Arrowheads show CHOP⁺ cells (pink/white nuclei). Scale bar, 20 μm. (B) Morphometry of transfected COS-7 cells showing transfection efficiency (bars i and iv) and PLP1⁺/CHOP⁺ cells expressed as a proportion of transfected (ii and v ) or stressed (iii and vi) cells. (C) Northern blots show induction of UPR effector genes in 293T cells untreated (NT), treated with tunicamycin (Tunic), or transfected with wild-type or mutant PLP1. (D) Oligodendrocyte progenitors from wild-type mice (Da) elaborate cell processes, differentiate, and incorporate Plp1 gene products into the plasma membrane (green). Progenitors from msd mice also extend processes, which are subsequently resorbed (arrows) after the cells differentiate and express the Plp1 gene (Db). These cells localize CHOP (red) in the nucleus which appears as yellow/orange (arrowheads). Scale bar, 10 μm.

Figure 2. Temporal Induction of Chop and Atf3 in Spinal Cord from Mutant Mice Correlates with Plp1 Expression

(A) Northern blots from wild-type (wt) and msd mice between P4–P21 express Plp1, Chop, and Atf3. Ethidium bromide-stained 18S rRNA serves as loading controls. (B) Northern blots from wild-type (wt), jp, and rsh mice at P18 and P21 show that Chop and Atf3 are elevated in the mutants. (C) Northern blots from msd, jp, and rsh mice show induction of molecular chaperones. (D) Northern blots from optic nerve of msd mice show induction of UPR effector genes (E) Northern blots from tunicamycin-treated 3T3 cells (2 μg/mL, 4 hr), wild-type, and msd mice show that Doc genes are not induced by mutant Plp1 expression. (F) Doc1 expression cannot be detected by nonquantitative RT-PCR from wild-type or msd mice after 35 cycles. Expression of Docs4 and 6 is detected. (G) Ribonuclease protection assays show that Doc4 expression is decreased in msd mice to 57% of controls. (H) Northern blots show that Doc4 transcripts are processed differently in wild-type and msd mice.

Figure 3. CHOP Is Localized to Nuclei of DM-20/PLP1⁺ Oligodendrocytes in P18 Cerebellum from msd and rsh Mice

(A) Immunofluorescence labeling in wild-type mice using anti-DM-20/PLP1 antibodies. (B) CHOP is not detected in this field. (C) DAPI stain shows locations of nuclei. (D) DM-20/PLP1 staining in msd mice is largely perinuclear (arrowheads). (E and F) Nuclei are stained with anti-CHOP antibodies (arrowheads). (G) Anti-DM-20/PLP1 antibodies label rsh myelin sheaths in the pons. (H and I) Oligodendrocyte nuclei are labeled with anti-CHOP antibodies (arrowheads). Inset shows an oligodendrocyte at high magnification with CHOP in the nucleus (arrowhead). Scale bars, 20 μm in (B); inset, 3 μm.

Figure 4. ATF3 Is Localized to Nuclei of DM-20/PLP1⁺ Oligodendrocytes in Brainstem from msd and rsh

(A) Immunofluorescence labeling of white matter from wild-type mice using anti-DM-20/PLP1 antibodies. (B) ATF3 is not detected in this field. Scale bar, 20 μm. (C) DAPI stain shows locations of nuclei. (D) DM-20/PLP1 staining in msd mice is largely perinuclear (arrowheads). (E and F) Nuclei in some oligodendrocytes are stained with anti-ATF3 antibodies (white arrowheads); others are unstained (black arrowheads). (G) Anti-DM-20/PLP1 antibodies label oligodendrocytes (arrowheads) and myelin sheaths in rsh mice. (H and I) Some cells are labeled with anti-ATF3 antibodies (white arrowheads), while others are ATF3⁻ (black arrowheads). Arrows show ATF3⁺, DM-20/PLP1⁻ cells.

Figure 5. Oligodendrocytes and Microglia Comprise the CHOP⁺ and ATF3⁺ Cell Populations in Brains of msd and rsh Mice

Essentially all CHOP⁺ cells in the CNS of msd and rsh mice are oligodendrocytes. Oligodendrocytes and microglia are the major cell populations comprising ATF3⁺ cells.

Figure 6. CHOP and ATF3 Are Induced in White Matter from a PMD Patient Harboring the Δexon6 Mutation

(A) Immunofluorescence labeling of mutant PLP1 (Aa) and DM-20 (Ab) encoded by cDNAs from a Δexon6 PMD patient show protein accumulating in the ER of transfected COS-7 cells. Scale bar, 5 μm. (B) Northern blot of white matter (WM) and gray matter (GM) from a multiple sclerosis patient (MS) and a Δexon6 PMD patient. 18S serves as loading controls. (C) An oligodendrocyte (arrow) labeled with anti-MBP antibodies (Ca) expresses CHOP (Cb) in the nucleus (Cc). Three additional oligodendrocytes (arrowheads and inset) express CHOP in the cytoplasm. Scale bar, 5μm.

Figure 7. Kaplan-Meier Analysis and Oligodendrocyte Apoptosis in Chop Null/rsh Mice

(A) Survival over 20 weeks in sib cohorts of wild-type, rsh, Chop null, Chop null/rsh, and Chop-het/rsh mice. The ordinate indicates the number of mice in each cohort. (B) Levels of apoptosis are 5-fold higher in cervical spinal cord from P16 msd mice compared to controls. (C) Electron micrographs show (Ca) a morphologically normal oligodendrocyte from a Chop null mouse surrounded by myelinated fibers and (Cb) an apoptotic oligodendrocyte from a Chop null/rsh mouse with condensed chromatin (arrowhead) surrounded by large unmyelinated axons (asterisks). Scale bar, 1μm.

Figure 8. Possible Models to Account for the Correlation between Disease Severity in PMD and DM-20/PLP1 Trafficking Defects

(A) UPR portrayed as a graded response that is proportional to the level of misfolded protein in the ER; the higher the level, the stronger the UPR. (Aa) Mutations causing ER accumulation of both PLP1 and DM-20 induce a strong UPR. (Ab) Mutations causing accumulation of PLP1 but not DM-20 induce a weak response. (B) UPR portrayed as an off/on switch upon misfolded protein accumulation. (Ba) PLP1 and DM-20 accumulation induces the UPR. DM-20 is toxic and disrupts cell survival pathways (?). (Bb) Accumulation of only PLP1 in the ER induces the UPR but does not affect cell survival pathways.