Supplementary Figure S1
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1 Supplementry Figure S1 d MAP2 GFAP e MAP2 GFAP GFAP c f Clindin GFAP Supplementry Figure S1. Neuronl deth nd ltered strocytes in the rin of n ffected child. Neuron specific MAP2 ntiody stining in the hippocmpus () nd cortex (), clindin ntiody stining of Purkinje cells in the cereellum (c) nd strocyte specific GFAP ntiody stining in the hippocmpus (d), cortex (e) nd cereellum (f) of control nd ffected children. stining in lue identifies cell nuclei. Scle rs= 25 µm. 1
2 Supplementry Figure S2 +Sudn Blck Lipofuscin Bright field Merge Bright field Merge Supplementry Figure S2. Confirmtion of lipofuscin ggregtes in ASMko mice rins y Sudn Blck B quenching nd right field microscopy. () Autofluorescence of lipofuscin ggregtes in the hippocmpus of 4-month-old ASMko mice ws quenched y Sudn lck B incution. () Bright field microscopy imges of wt nd ASMko mouse hippocmpus evidence undnt microgrnulr refringent deposits, comptile with lipofuscin ggregtes, in the ASMko mice. stining in lue identifies cell nuclei. Scle rs = 10 µm. 2
3 Supplementry Figure S3 ** ** Lysenin c SM 16h SM 24h d Lysenin Cont SM 16h Cont SM 24h SM GW4869+Sir+SM * SM levels (.u.) *** Lysenin SM GW+Sir+SM 3
4 Supplementry Figure S3. SM levels in ASMko hippocmpl extrcts nd cultured neurons nd in wt cultured neurons incuted with the lipid. () Grph shows men ± SEM of SM levels in nmol/mg protein in hippocmpl extrcts from 4- month-old wt nd ASMko mice (n = 5, Student s t-test, P = 0.001). (, c, d) Lysenin stining tht detects SM in 12 DIV non-permeilized hippocmpl neurons from wt nd ASMko mice () or in neurons from wt mice treted or not with SM for 16 nd 24 hours (c) or with SM in the presence of the neutrl nd cid sphingomyelinse inhiitors GW4869 nd sirmesine, respectively (d). Scle rs = 10 µm. Grphs show men ± SEM of Lysenin ssocited fluorescence in ritrry units (n = 70 neurons from 3 independent cultures, Mnn-Whitney U-test, P = in ; Student s t-test, P 16h = nd P 24h = in c; two-wy ANOVA followed y Gmes-Howell, P CTL-SM = ; P CTL-GWSIR = 0.006; P SM-GWSIR = in d). 4
5 Supplementry Figure S4 Supplementry Figure S4. Dily wter consumption in wt nd ASMko mice treted or not with complexed with HOP-β-CDX or with HOP-β-CDX only. Grph shows the verge mount of wter consumed per mice in ml per dy in ech week of the tretment (right pnel) or s n verge mount during the whole 6 weeklong tretment (left pnel). No significnt differences were oserved in wt or ASMko mice treted only with HOP-β-CDX or with complexed with HOP-β-CDX (). 5
6 Supplementry Figure S5 Ac-H3K18 H3 Actin Supplementry Figure S5. Orl tretment with ffects histone decetyltion ut not SM levels in the mouse hippocmpus. () Grph shows men ± SEM of SM levels in nmol/mg protein in hippocmpl extrcts from wt nd ASMko mice orlly treted or not with (n = 7, two-wy ANOVA followed y Gmes Howell, P wt/wt- = 0.998, P ko/ko- = 0.754, P wt/ko = , P wt-/ko- = 0.001). () Western lot nlysis of cetylted histone 3 (Ac-H3K18), totl histone 3 (H3) nd ctin in hippocmpl extrcts from wt nd ASMko mice orlly treted or not with. Grphs show men ± SEM of cetylted histone 3 levels (n = 7, Student s t-test, P wt = 0.023; P ko = 0.019). 6
7 Supplementry Figure S6 Untreted +HOP-β-CDX Untreted +HOP-β-CDX PMCA ATP6V1A ROS levels (.u.) ROS levels (.u.) Untreted HOP-β-CDX HOP-β-CDX + Untreted HOP-β-CDX HOP-β-CDX + Supplementry Figure S6. Tretment with HOP-β-CDX does not ffect PMCA nd ROS levels in the rin of wt nd ASMko mice. () PMCA nd ATP6V1A Western lot in hippocmpl memrne extrcts from wt nd ASMko mice untreted or treted with HOP-β-CDX. Grphs show men ± SEM of PMCA levels normlized to ATP6V1A (n = 7, Student s t-test, P wt = 0.094, P ko = 0.834). () Grphs show men ± SEM of the fluorescence ssocited to DHR tht detects ROS levels in the hippocmpus of wt nd ASMko mice untreted, treted with HOP-β- CDX only or with complexed with HOP-β-CDX (n = 7, two-wy ANOVA followed y Gmes Howell). 7
8 Supplementry Figure S7 Frctin Frctin positive cells / mm 2 Cleved cspse-3 19 kd 17 kd GAPDH Cleved cspse-3 (.u.) Supplementry Figure S7. Neuronl deth does not tke plce in the hippocmpus of 4 month-old ASMko mice treted or not with. () Representtive imge of frctin positive cell in the hippocmpus. Dpi in lue stins cellulr nuclei. Scle r = 5 µm. Grph shows men (± SEM) numer of frctin positive cells per re unit in the hippocmpus of wt nd ASMko mice (n = 6, Mnn-Whitney U-test, P = 0.686). () Western lot of the 19 nd 17 kd frgment derived from cspse-3 clevge, indictive of poptosis, nd of GAPDH used s loding control in hippocmpl extrcts of wt nd ASMko treted or not with. The grph shows the men (± SEM) cspse-3 frgment levels normlized to GAPDH (n = 7, two-wy ANOVA followed y minimum significnt difference, P wt-wt+sh = 0.426, P wt-ko = 0.278, P ko-ko+sh = 0.243). 8
9 Supplementry Figure S8 c I/II I/II III III IV/V IV/V I/II III IV/V I/II III IV/V Clindin VI VII VIII VI VII VIII VI VII VIII VI VII VIII IX X IX X IX X IX X 9 Clindin Clindin
10 Supplementry Figure S8. Purkinje cell survivl in cereellr loes of ASMko mice. Clindin immunostining of Purkinje cells in nterior I-V (), mid VI-VIII () nd posterior IX-X (c) loes of the cereellum of control or treted wt nd ASMko mice. Grphs show men ± SEM of numer of clindin positive cells per re unit (n = 7, two-wy ANOVA followed y Gmes Howell, for nterior loes P wt/ko = 0.022, P wt/ ko sh = 0.024, P ko/ko-sh = 0.182; for mid loes P wt/ko = 0.016, P wt-sh/ ko = 0.036, P ko/ko-sh = 0.041; for posterior loes P wt/ko = 0.002, P wtsh/ ko = , P ko/ko-sh = 0.022). stining in lue identifies cell nuclei. Scle rs = 100 µm. 10
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