Supplementary Information

Therapy of Pelizaeus-Merzbacher-Disease in mice

with dietary cholesterol

Gesine Saher, Fabian Rudolphi, Kristina Corthals, Torben Ruhwedel, Karl-Friedrich

Schmidt, Siegrid Löwel, Payam Dibaj, Wiebke Möbius, and Klaus-Armin Nave

Supplementary Item Title Supplementary Figure 1 Influence of cholesterol diet

Supplementary Figure 2 Impaired blood brain barrier integrity

Supplementary Figure 3 Optomotoric analysis

Supplementary Figure 4 PLP and cholesterol in lysosomes

Supplementary Figure 5 Cholesterol diet reduces accumulated PLP

Supplementary Figure 6 Histological analysis of the spinal cord

Supplementary Figure 7 Hypomyelination and enhanced intracellular PLP deposits in Plp1 transgenic mice lacking cholesterol biosynthesis in oligodendrocytes

Supplementary Figure 8 Subcellular distribution of cholesterol

Supplementary Table 1 PMD patients and mouse models

Supplementary Video 1 Motor performance of treated and untreated PMD mice. This video shows an example of a beam test that was used to evaluate motor abilities of PMD mice.

Supplementary Video 2 Motor disabilities of cSQS KO and cSQS KO*PLP+/66 mice.

Nature Medicine doi:10.1038/nm.2833

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Supplementary Figure 1: Influence of cholesterol dieta) PMD mice have normal serum cholesterol levels before treatment at P30 (wt 105 +/- 2; PMD normal diet 112 +/- 8; PMD that will receive chol diet 101 +/- 2 mg/dl). When on cholesterol diet, the serum cholesterol levels increase slightly but significantly (113 +/- 5 wt; 116 +/- 2 PMD normal diet; 127 +/- 1 PMD chol diet; n = 4-6 mice).b) Even after prolonged time on cholesterol diet, PMD mice show comparable body weight (n=4-5).

Nature Medicine doi:10.1038/nm.2833

Supplementary Figure 2: Impaired blood brain barrier integritya) Evans blue derived fluorescence in brain is restricted to blood vessels in wild type mice (negative control), but extravasates in ApoE null mice (positive control) and PMD mice disregarding the diet. Capillaries with extravasating Evans blue are marked by white arrowheads, normal capillaries by open arrowheads. b) Bodipy-cholesterol derived fluorescence is detectable in myelin of the sciatic nerve of wild type mice one week after i.p. injection (arrowhead, node of Ranvier). Scale, 10μm.

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Supplementary Figure 3: Optomotoric analysisContrast sensitivity and drift speed were measured before and after treat-ment on wt (n=4) and PMD mice (n=9 per group).

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Spatial frequency (cyc/deg)

Nature Medicine doi:10.1038/nm.2833

Supplementary Figure 4: PLP and cholesterol in lysosomes Immunoelectron microscopic image reveals cholesterol (BCtheta) and PLP in oligodendroglial lysosomes of optic nerve from PMD mice at 2 weeks of age (scale 100 nm; lysosome, ly).

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Nature Medicine doi:10.1038/nm.2833

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Supplementary Figure 5: Cholesterol diet reduces accumulated PLPa) Immunolabeling of PLP and Lamp1 on spinal cord from WT and PMD mice revealing a strong reduction of the endo/lysosomal compartment and a substantial increase in myelination in cholesterol treated animals. Nuclei were stained with DAPI (scale, 20 μm).b) Spinal cord sections from WT and PMD mice (chol, cholesterol diet from 2 to 12 weeks of age) were stained for PLP and activated microglia (MAC3). Nuclei are stained with DAPI. While microglia may contain some PLP (arrowheads), cells with extended perinuclear PLP accumulation (asterisk) are not MAC3 positive microglia (cell outline marked by dashed line). Scale, 10 μm.

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Nature Medicine doi:10.1038/nm.2833

Supplementary Figure 6: Histological analysis of the spinal corda) Electron microscopic images of the corticospinal tract (CST) of PMD mice on normal or cholesterol diet, in comparison to age matched wild type mice.b) Quantification of CC-1 positive cells in the CST. In the CST area the number of nuclei were counted that were directly surrounded by CC-1 derived signal (n=3 animals).c) Quantification of myelin content in the CST via G-ratio measurement (n=3 animals).d) Mean axon caliber (and caliber distribution, not shown) is comparable in the CST of PMD mice and wild type mice.e-f) Immunodetection of microglia (MAC3) (e) and astrocytes (GFAP) (f) on spinal chord sections of PMD mice.g) Counting MAC3 positive microglia and GFAP positive astrocytes in the CST shows dramatic reduction of gliosis in cholesterol fed PMD mice. In the CST area the number of nuclei were counted that were directly surrounded by MAC3 (microglia) or GFAP (astrocytes) derived signal (n=7-9 animals per group).h) Immunolabeling of CD3 positive T cells (anti CD3) with quantification in the dorsal funiculus (n=4-7). Scale, 5 μm (a), 50 μm (e, f, h); *, p<0.05; **, p<0.01; ***, p< 0.001.

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Nature Medicine doi:10.1038/nm.2833

Supplementary Figure 7: Hypomyelination and enhanced intracellular PLP deposits in Plp1 transgenic mice lacking cholesterol biosynthesis in oligodendrocytesSince providing cholesterol to PMD oligodendrocytes reduces the abnormal storage of PLP in lysosomes, a decrease of cholesterol availability might aggravate PMD pathology. Conditional SQS/Cnp-cre mutants (cSQS KO) that overexpress Plp1 only mildly (PLP-tg+/66)1 (cSQS KO * PLP-tg+/66) were compared with WT mice, cSQS KO, and PLP overexpressing mice (PLP-tg+/66). PLP-tg+/66 mice do not show PMD symptoms before four months of age2, 3, 4. Immunofluorescence detecting PLP and Lamp1 on spinal cord of P100 animals reveals PLP accumulation in Lamp1 positive compartments predominantly in cSQS KO * PLP-tg+/66 mice (marked by arrows). Limited cholesterol in oligodendrocytes worsens the pathology of PLP overexpression in mice and increases the probability of its accumulation in endo/lysosomes. (N, neuronal cell body; scale, 10 μm).

1. Readhead,C., Schneider,A., Griffiths,I., & Nave,K.A. Premature arrest of myelin formation in transgenic mice with increased proteolipid protein gene dosage. Neuron 12, 583-595 (1994).2. Anderson,T.J. et al. Distinct phenotypes associated with increasing dosage of the PLP gene: implications for CMT1A due to PMP22 gene duplication. Ann. N. Y. Acad. Sci. 883, 234-246 (1999).3. Anderson,T.J. et al. Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene. J. Comp Neurol. 394, 506-519 (1998).4. Ip,C.W. et al. Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes. J. Neurosci. 26, 8206-8216 (2006).

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Nature Medicine doi:10.1038/nm.2833

Supplementary Figure 8: Subcellular distribution of cholesterol a) Primary Plp-tg oligodendrocytes were cultivated for 5 days in differentiation media with or without cholesterol. Cells were stained for cholesterol (filipin, white), endoplasmic reticulum (PDI, red), and endo/lysosomes (Lamp1, green). Approximate localization of the nuclei is outlined (dashed line). In Plp-tg oligodendrocytes grown in the absence of cholesterol supplementation, filipin can be found intracellularly exceeding the endo/lysosomal compartment (arrowheads). When cells were grown in the presence of cholesterol, filipin only rarely stains structures in the cell soma but strongly labels the plasma membrane (scale 10μm).b) Electron micrographs stained for cholesterol (BCtheta, 10nm gold) revealed that the ER cisternae (asterisk) contain increased amount of cholesterol in 2 weeks old PMD mice in comparison to wild type controls (n, nucleus; g, Golgi).

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Nature Medicine doi:10.1038/nm.2833

Supplementary table 1 PMD patients and mouse models

Pathology

Onset of symptoms

Life expectance

Blood brain barrier

Plp1-tg72/72

dysmyelination, followed by demyelination and inflammation (~1 month), at 85-120d almost complete absence of myelin; severe motor impairment, reduced

conduction velocity in optic nerves (40d)6-10

~1 month*,6,10 premature death(120 ± 2 days)* leaky*

Plp1-tg+/66 normal development,

late onset demyelination and axonal degeneration6-8 >10 months7,8 >18 months8 normal*,8,17

PMD patients PLP1 duplication

hypomyelination, impaired motor development, nystagmus, ataxia, psychomotor deterioration, cognitive

impairment; less commonly seizures, choreoathetosis1,2

within first months

infancy up to adulthood

?

* this study

Nature Medicine doi:10.1038/nm.2833