Supporting Information

Transcription

1 Supporting Information Unno et al /pnas SI Methods Protein Expression. Protein extracts were obtained by using NE- PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). Forty micrograms of protein was loaded on SDS gel. PVDF blots were probed with anti-mouse Ca v 2.1 antibody (1:500), anti-human Ca v 2.1 antibody (A6RPT 1:10,000), or 1C2 (1:10,000). Immunohistochemistry. Formalin-fixed, paraffin-embedded sections in 4-μm thickness were deparafinized. For antigen retrieval, sections were deparaffinized with xylene, washed with distilled water, and boiled for 20 min in 10 mm citrate buffer (ph 7.4) to detect PC. For Ca v 2.1 and Cathepsin immunohistochemistry, sections were immersed in formic acid for 6 min. After antigen retrievals, all sections were washed in distilled water, treated with 0.3% (vol/ vol) hydrogen peroxide in methanol to quench endogenous peroxide, and then incubated with a mixture of normal goat and horse sera for 30 min. Finally, sections were incubated overnight at 4 C with anti-ca v 2.1 antibody (A6RPT-polyQ 1:1,000), 1C2 (Millipore; 1:1,000), Cathepsin D (Abcam; 1:1,000), and Calbindin D-28k (Swant; 1:1,000). The primary antibodies were serially detected with the appropriate biotinylated anti-rabbit or anti-mouse IgG (Vector), avidin-biotinylated-peroxidase complex (Vector), and finally developed with 3,3 -diaminobenzidine and hydrogen peroxide as chromogen. Between each step, sections were washed with PBS containing 0.05% Tween 20 three times. For double immunofluorescent labeling, sections were similarly treated and incubated with primary antibodies overnight. After washing with PBS, primary antibodies were detected with Alexa Fluor 488 conjugated anti-rabbit goat IgG (Invitrogen) and Indocarbocyamine conjugated anti-mouse horse IgG (Jackson ImmunoReserch; both diluted at 1: 1,000) for 1 h at room temperature. Sections were then washed and examined under a confocal light microscope (TCS-NT, Leica; LSM 510META, Carl Zeiss). Quantification of the cytoplasmic areas showing coimmunofluorescence for each Purkinje cell (PC) was performed with the Win Roof version 5 (Mitani). TUNEL Assay. For the TUNEL assay, we removed the paraffin from sections and carried out the TUNEL reaction in accordance with the manufacturer s instructions (in situ Cell Death Detection kit; Roche). Preparation of Acutely Dissociated PCs. PCs were freshly dissociated from 28- to 42-d-old WT and the two kinds of KI mice. Coronal slices (350 μm) of the cerebellum were prepared by using a microslicer (Linear Slice PRO7; Dosaka). After preincubation in Krebs solution for 20 min at 34 C, the slices were digested: first in Krebs solution containing 0.02% Pronase (Calbiochem/Novabiochem) for 15 min at 34 C, and then in solution containing 0.02% thermolysin (typex; Sigma Aldrich) for 15 min at 34 C. The Krebs solution used for preincubation and digestion contained the following: 124 mm NaCl, 5 mm KCl, 1.2 mm KH 2 PO 4, 2.4 mm CaCl 2, 1.3 mm MgSO 4, 24 mm NaHCO 3, and 10 mm glucose. The solution was continuously oxygenated with 95% O 2 and 5% CO 2. PCs were dissociated mechanically by using fine glass pipettes with a tip diameter of μm. Dissociated cells settled on tissue culture dishes (Primaria no. 3801; BD Biosciences) within 30 min. Whole-Cell Voltage Recording. Currents were recorded at room temperature (25 28 C) by using the whole-cell mode of the patch-clamp technique with an EPC-10 patch-clamp amplifier (HEKA). Patch pipettes were made from borosilicate glass capillaries (1.5-mm outer diameter; Hilgenberg) by using a model P-97 Flaming-Brown micropipette puller (Sutter Instrument). Pipette resistance ranged from 2 to 3 MΩ when filled with the pipette solutions described below. Series resistance was electronically compensated to >50%, and both the leakage and the remaining capacitance were subtracted by -P/6 method. Currents were sampled at 10 khz after low-pass filtering at 2.9 khz. Ba 2+ currents were recorded in an external solution that contained the following: 150 mm tetraethylammonium chloride, 2 mm BaCl 2, 10 mm glucose, and 10 mm Hepes, ph adjusted to 7.4 with tetraethylammonium hydroxide. The pipette solution contained the following: 85 mm CsOH, 85 mm aspartate, 40 mm CsCl, 4 mm MgCl 2, 5 mm EGTA, 10 mm Hepes, 2 mm disodium ATP, and 8 mm creatine phosphate, ph adjusted to 7.2 with CsOH. Rapid application of drugs was made by a modified Y-tube method. The external solution surrounding the recorded cell was completely exchanged within 200 ms. Slice Preparation. Firing pattern of the cerebellar Purkinje cells were measured using 300-μm-thick parasagittal cerebellar slices from 38- to 41-d-old mice. Slices were cut in ice-cold solution using the microslicer (Pro-7; Dosaka EM). They were incubated at 34 C for 30 min for recovery and thereafter maintained at room temperature. The solution used for slicing contained the following: 80 mm NaCl, 25 mm NaHCO 3, 25 mm glucose, 2.5 mm KCl, 1.25 mm NaH 2 PO4, 0.5 mm CaCl 2,3mMMgCl 2,and85mM sucrose at ph 7.4 when bubbled with 95% O 2 and 5% CO 2. Electron Microscopic Analysis. Mice were intracardiacally perfused with 2% (vol/vol) paraformaldehyde and 1% (vol/vol) glutaraldehyde and processed for transmission electron microscopy by using standard procedures. Immunoelectron Microscopy Using Ultrathin Cryosections. Ultrathin cryosections were prepared as reported (1). Briefly, anesthetized mice were fixed by cardiac perfusion using 4% paraformaldehyde buffered with 0.1 M PB (ph 7.2). Brain tissues containing cerebellum were excised from the mice, further immersed in the same fixativeat4 Cfor2h,embeddedin12%gelatinin0.1MPB(pH 7.2). Small blocks were rotated in 2.3 M sucrose in PB overnight at 4 C and quickly plunged into liquid nitrogen. Sections 60-nm thick were cut with a Leica UC6/FC6 or UC7/FC7 ultramicrotome and picked up with a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose. The sections were reacted overnight at 4 C with rabbit anti-lamp1 (Abcam; 1:10) and then for 1 h at RT with goat antirabbit IgG conjugated with 10-nm colloidal gold particles (British Biocell International), and examined with a Hitachi H-7100 electron microscope. For control experiments, ultrathin sections were reacted only with the gold particle-conjugated secondary antibody. Activity Assay of Cathepsins B and D. Whole homogenetes of cerebellum from 9-wk-old WT and ctsb / mice were used to analyze the activity of cathepsins B by using SensoLyte 520 Cathepsin B assay kit (ANASPEC) and cathepsin D by using cathepsin D Assay Kit (Sigma) according to the kit instructions. 1. Koike M, et al. (2000) Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons. J Neurosci 20(18): of10

2 Fig. S1. Generation of Sca6- KI mice. (A) Germ-line transmission of an 118Q allele revealed by Southern blot analysis using 5 probe shown in Fig. 1A. (B) Western blot analysis performed on nuclear fraction of cerebellar lysates from 6-wk-old WT, 11Q/11Q, 118Q/118Q, 11Q/+, and 118Q/+ mice, all blotted against anti-ca v 2.1 antibodies raised against intracellular loop between domains II and III (Upper) and A6RPT-polyQ (Lower) antibodies. (C) Footprints of 15-wk-old mice. Fp and Hp mean front paws and hind paws, respectively. 2of10

3 A wild-type 11Q/11Q 118Q/118Q B wild-type 30 ms 11Q/11Q 40 mv -50 mv -80 mv 118Q/118Q D wild-type 11Q/11Q 118Q/118Q -Aga IVA -Aga IVA -Aga IVA control control control C Current density (pa/pf) Membrane potential (mv) wild-type 118Q/118Q 11Q/11Q E Relative current wild-type 118Q/118Q 11Q/11Q τ=18.5, I base =0.03 τ=15.4, I base =0.03 τ=20.0, I base = Time (s) Fig. S2. Whole-cell Purkinje Ba 2+ currents recorded in acutely dissociated PCs. (A) Confocal images of PCs, dissociated from 7-wk-old wild-type, 11Q/11Q,or 118Q/118Q mouse and immunolabeled with the anti-ca v 2.1 Abs raised against intracellular loop between domains II and III. Lower indicates DIC images. The localization of Ca V 2.1 at plasma membrane was indistinguishable among the three groups. Arrows mark out PCs. (Scale bar: 10 μm.) (B) Families of Ba 2+ currents evoked in P28 32 PCs by 30-ms step depolarization pulses from 50 to 40 mv with 10-mV increments from a holding potential (V h )of 80mV. (C) Current density voltage relationships for P28 32 wild-type (open circle, n =10), 118Q/118Q (filled circle, n = 10), and 11Q/11Q (filled triangle, n = 16) mice. Each point represents average value, and error bar shows mean SEM if they are larger than symbols. Curves were fitted with the equation, I (V m )=G (V m E rev )/(1 + exp((v 0.5 V m )/k), where I (V m ) is the peak Ba 2+ current at the membrane potential of V m, G is the maximum conductance, E rev is the apparent zerocurrent potential in the I V relationship, V 0.5 is the potential to give a half-value of conductance, and k is the slope factor that determines the steepness of the curve. The values of E rev, V 0.5,andk, are 39.7 mv, 27.1 mv, and 3.79 mv for wild-type, 39.1 mv, 26.2 mv, and 3.96 mv for 118Q/118Q, and 39.1 mv, 27.2 mv, and 3.86 mv for 11Q/11Q KI mice. (D) Blockade of Ca 2+ channel by 100 nm ω-aga IVA. Ba 2+ currents were evoked by the 30-ms step pulse to 10 mv from a V h Legend continued on following page 3of10

4 of 80 mv. (E) Time courses of peak Ba 2+ current amplitudes in response to application of 100 nm ω-aga IVA for wild-type (open circle, n = 5), 118Q/118Q (filled circle, n = 5), and 11Q/11Q (filled triangle, n = 4) KI mice. The Ba 2+ current amplitudes reduced to 4.1 ± 1.1% for wild-type, 2.8 ± 0.9% for 118Q/118Q, and 2.2 ± 0.4% for 11Q/11Q KI mice. Each point represents average value, and error bar shows mean SEM if they are larger than symbols. Curves were fitted by a single exponential function with time constant of 18.5 s, 15.4 s, and 20.0 s for wild-type, 118Q/118Q, and 11Q/11Q KI mice, respectively. Fig. S3. Firing patterns of PCs in slice preparations from 40-d-old WT (A C) and 118Q/118Q KI mice (D). (A) Na + spikes in a wild-type PC evoked by injecting 1.5-s depolarizing current (1,000 pa). (B) Oscillatory response of Na + and Ca 2+ spikes in the wild-type PC evoked by injection of depolarizing current (1,500 pa). Four neurons of 18 wild-type neurons (from P =39 41) exhibited Ca 2+ spikes. (C) Na + spikes were terminated in the wild-type PC during the depolarizing current injection (1,500 pa) in the presence of 50 μm Cd 2+ in the extracellular solution. (D) Na + spikes were terminated in a PC from 118Q/118Q KI mice even during the current injection (1,500 pa). None of Q-neurons (from P =38 40) exhibited Ca 2+ spikes. All electrical recordings were done from PCs located in anterior lobe. 4of10

5 Fig. S4. Pathological analysis of -118Q and -11Q KI mice. (A) Tunel assay (Left) and cleaved caspase-3 (Right) immunohistochemistry showed apoptotic PC death in the cerebellum of a 10-wk-old 118Q/118Q mouse. (B) Anticalbindin staining of 14-mo-old WT and 11Q/11Q mice failed to show any significant changes in the PC body and dendrite. (C) Immunohistochemical analysis using A6RPT-polyQ antibody show cytoplasmic inclusion formation in 118Q/+ cerebellum. (D) Double immunofluorescense analysis using calbindin (red) and A6RPT-polyQ (green) indicates cytoplasmic and dendritic inclusion formation in the PC of a 52-wk-old 118Q/+ mouse. (E) Immunohistochemical analysis using A6RPT-polyQ antibody on cross-sections of WT and 11Q/11Q cerebellum at 14 mo of age. (Scale bars: B Left, 1 mm; B Right, C, and D, 20μm.) 5of10

6 118Q/118Q 118Q/118Q 118Q/118Q Fig. S5. Characterization of neuronal inclusions in 118Q/118Q PCs. (A) Immunohistochemical analysis using antiubiquitin antibody failed to detect inclusion formation in PCs of a 25-wk-old 118Q/118Q mouse. (B) Double immunofluorescence microscopy for A6RPT-polyQ and KDEL (red). DAPI was used for nuclear staining (aqua blue). (C) Double immunofluorescence microscopy for 1C2 (red) and Tom20 (green). DAPI was used for nuclear staining (aqua blue). (Scale bars: A, 100μm; B and C, 20 μm.) Fig. S6. CHOP expression in the cerebellum of Sca6- KI mice. (A) Representative immunohistochemical staining for CHOP on the cerebellum sections of 118Q/118Q mice and their WT littermates at 9 wk of age. (B and C) Expression levels of CHOP in cerebellar extracts from 9-wk-old WT, 11Q/11Q,and 118Q/118Q mice (n = 3, each). B shows an image for Western blot analysis and the graph in C represents the mean ± SEM of CHOP expression levels, normalized to those of β-tubulin. 6of10

7 Fig. S7. Morphological analysis of aged Sca6 84Q/84Q mice. (A) Electron microscopic analysis of a 1-y-old Sca6 84Q/84Q mouse. Large electron dense structrure was observed in the cytoplasm of a PC. (B) Immunohistochemical analysis using anticalbindin antibody on cerebellar sections obtained from a 30-wk-old Sca6 84Q/84Q mouse. (C) Immunohistochemical analysis using anticalbindin antibody on cerebellar sections obtained from a 30-wk-old Sca6 84Q/84Q ; ctsb / mouse. (Scale bars: ALeft,2μm; A Right, 500 nm; BLeftand CLeft, 100 μm; B Right and CRight,50μm.) 7of10

8 Fig. S8. Analysis of autophagic response in 118Q/118Q mice. (A) Western blot analysis using anti-lc3 antibody performed on cytoplasmic fraction of brain extracts from 7-wk-old wild-type (WT) and 118Q/118Q mice. (B) Expression levels of phospho-s6 ribosomal protein in cerebellar extracts from 7-wk-old WT, 11Q/11Q, and 118Q/118Q mice. (B Left) A representative image of Western blots probed with antibodies specific for phospho- (p-) and total S6 ribosomal protein. (B Right) The relative levels of phospho-s6 (p-s6) to total S6 (n = 3 for each group). Values were determined by densitometric scanning. Error bars indicate SEM. (C) Immunohistochemical analysis using anti-p-s6 (Ser235/236) antibody on cerebellar sections. (D) Immunohistochemical analysis using anti-lc3 antibody (red) on liver (Upper) and cerebellar sections (Lower). Liver sections were stained with Alexa 488 phalloidin to visualize cell outlines (green), and DAPI was used for nuclear staining. The animals were fed either normally (Normal-fed) or food-restricted for 48 h (Food-rest 48 h) before euthanization to induce autophagy in the liver. 8of10

9 Fig. S9. Characterization of ctsb / mice. (A) Immunohistochemical analysis using anticalbindin antibody on crerebellar sections of a 30-wk-old ctsb / mouse. (Scale bar: Left, 1 mm; Right, 20μm.) (B) Cathepsin B activity in 9-wk-old WT and ctsb / cerebella (n = 3, each). Error bar means SEM. (C) Cathepsin D activity in 9-wk-old WT and ctsb / cerebella (n = 4, each). Error bar means SEM. Fig. S10. Immunohistochemistry of cerebellar sections from 4-wk-old Sca6 118Q/118Q ; ctsb +/+ and Sca6 118Q/118Q ; ctsb / mice. Sections were stained with anticalbindin (A and C) or A6RPT-polyQ (B and D) antibody. 9of10

10 Movie S1. Cage behavior of 3-mo-old WT, 11Q/11Q, and 118Q/118Q mice. Movie S1 10 of 10