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1 Supporting Online Material for Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy Qian Yang, Hua She, Marla Gearing, Emanuela Colla, Michael Lee, John J. Shacka, Zixu Mao* *To whom correspondence should be addressed. Published 2 January 2009, Science 323, 124 (2008) DOI: /science This PDF file includes: Materials and Methods Figs. S1 to S9 References
2 Material and Methods Plasmid, antibodies and chemicals: pcdna/hygro-antisense-lamp2a and pcdna/zeo(-)-antisense-hsc70 were gifts from Janice S. Blum (1), and Lamp2a construct from A.M. Curevo. Antibodies were purchased from following vendors: anti- MEF2D and anti-α-synuclein from BD Biosciences, anti-hsc70 and anti-lamp2a from Abcam, and anti-vp16 and anti-gst from cell signaling Technology. NH4Cl, 6-AN and 3-MA were purchased from Sigma, LMB from LC Laboratories, and luciferase reporter gene assay kit and cell proliferation reagent WST-1 from Roche. Cell culture: SN4741 cells were cultured in DMEM (Gibco) containing 10%FBS (2). To remove the serum, cells were washed four times with PBS before replacing the complete medium. Primary cultures of cortical and cerebellar granule neurons were described previously (2-4). Isolation of lysosomes: Lysosomes were isolated as described (5). Male Long-Evans rats were fasted for hours before sacrifice. Livers were removed, washed with cold PBS and homogenized in the extraction buffer (Sigma, lysosomal isolation kit). After separation by density gradient centrifugation (150,000 g for 4 hours), lysosomes were isolated from lysosomel fraction and tested for Lamp2a levels by immunoblotting. The intactness of the lysosomes was assessed using the dye Neutral Red (Sigma, N2537). Binding and uptake by lysosomes: The assays were carried out as described (6). For binding assay, isolated lysosomes were co-incubated with purified GST-MEF2D fusion proteins for 20 minutes at 37 o C and washed for four times with cold PBS. For uptake assay, isolated lysosomes were treated with 100μM chymostation for 10 minutes on ice and incubated with the purified GST-MEF2D fusion proteins or cellular lysates for 20 minutes at 37 o C in MOPS buffer. Proteinase K was added directly to the samples for 10 minutes on ice.
3 A53T α-synuclein transgenic mouse and cathepsin D deficient mouse: The characterization of these mouse models has been described elsewhere (7, 8). Real-time PCR. Complementary DNA was generated from total RNA using random priming and MMLV reverse transcriptase (Invitrogen). Semi-quantitive PCR was done on Mycycler (Bio-rad) as following: 95 0 C,3 min; 30 cycles of 94 0 C,30 sec; 56 0 C,20 sec; 72 0 C, 25 sec; followed by 72 0 C,10 min. Real-time PCR was performed using SYBR Green PCR Master Mix (Invitrogen) and analysed on an AB Prism 7000 analyser (Applied Biosystems). All real-time values were normalized to 18S rrna. The oligonucleotides used in the study were: 18S forward, 5'- CGGCTACCACATCCAAGGAA-3', 18S reverse, 5'- GAGCTGG AATTACCGCGGCT-3'; MouMEF2D-F: gcagccacaaccgccacaacc, MouMEF2D-R: tgtgagagcagcacccacgtg. Electrophoretic mobility shift: Electrophoretic mobility shift assay was performed as described (4). The cell lysates were incubated with MEF2 specific probe or mutant probe. For super shift assay, the lysates were incubated with MEF2D antibody for 15 minutes before incubation with labeled probe. GST-pull down: SN4741 or HEK293 were washed in PBS and lysated in lysis buffer (20mM Tris ph 7.4, 40mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na3VO4, 10mM NaF, 10mM sodium pyrophosphate, 10mM sodium β-glycerophosphate, 1% Triton X- 100), and centrifuged. The supernatant was incubated with purified GST fusion proteins with glutathione sepharose beads (GE) for 2 hours at 4. Lusiferase reporter gene assay: MEF2 luciferase reporter assays were performed as described (4). SN4741 cells were transfected with a MEF2 luciferase reporter gene (wild type MEF2 binding site: TCGACGGGCTATTTTTAGGGCC; mutated binding site: 5- TCGACGGGCGATTTTTCGGGCCG-3) by Lipofectamine 2000 transfection method (Invitrogen). Cellular extracts were assayed for both luciferase and β-gal activities. Relative fold of luciferase activity was calculated based on efficiency of transfection.
4 Pulse-chase of MEF2D with 35 S Met/Cys: SN4741 cells grown to 60-70% confluence were washed and placed in Met/Cys-free media containing 10% dialyzed FCS. 320μCi of 35 S-label were added to cells for 12 hours. After removal of radioactive media, cells were washed and placed in regular media with or without FCS for various lengths of time. WST assay: This colorimetric assay measures the conversion of tetrazolium salt to formazan dye by cellular enzymes, which correlates to the number of metabolically active cells. SN4741 cells were cultured in 96 well microplates. After treatment, 10μl cell proliferation WST-1 reagent were added to each well when cells reached 60-70% confluence and incubated under conditions according to Manufacture s instruction (Roche). Statistical analysis: All statistical analyses where indicated are the Student s t test expect for fig. 3E and S7, which used rank sum test.
5 Supplementary figures and legends Fig. S1. Modulation of MEF2D levels in neurons. (A) Inhibition of MEF2D degradation by blocking autophagy. NH 4 Cl leads to MEF2D accumulation in primary cerebellar granule neurons (top panel: DIV indicates days in vitro) and primary cortical neurons (bottom panel:10mm NH 4 Cl). (B) Increase of MEF2D in the cortex of cathepsin D- deficient mice. Images show the immunohistochemical analysis of MEF2D in the cortical regions of wt and cathepsin D-deficient (CDKO) mice (red, MEF2D; blue, Dapi. Scale bar, 10μM). Tissue lysates from two sets of control and cathepsin D-deficient (CD-/-) mice were analyzed. The bottom graph shows relative MEF2D levels (n=3; * = p<0.05).
6
7 Fig. S2. The effects of serum deprivation on MEF2D transcript and protein. (A) Serum deprivation-induced turnover of metabolically labeled MEF2D. SN4741 cells were pulse-labeled with 35 S-Met/Cys for 12 hours. After removal of radioactive media, cells were treated with serum deprivation for the indicated times. MEF2D was immunoprecipitated and analyzed by autoradiography (top panel: MEF2D; bottom panel: actin levels in lysates). The bottom graph shows the quantitative change of MEF2D (numbers are mean±sem, n=3). (B) Semi-quantitative and quantitative RT-PCR analysis of mef2d mrna. Total RNA isolated from SN4741 cells following serum deprivation was analyzed by semi-quantitative (top: mef2d and 18S transcripts) and quantitative (bottom: levels of mef2d transcript relative to 18S transcript) RT-PCR.
8 Fig. S3. Effect of 3-MA on MEF2D level. SN4741 cells were treated with the macroautophagy inhibitor 3-MA for 12 or 24 hours. The levels of MEF2D were determined by immunoblotting. Bottom graph shows the quantification of 3-MA results (n=4).
9 Fig. S4. Lysosomal binding and uptake of RNase A. The purified known CMA substrate RNase A was incubated with purified lysosomes in uptake assays and detected by immunoblotting.
10 Fig. S5. Presence of multiple CMA recognition motifs in MEF2D. (A) Presence of multiple imperfect KFERQ-like sequences (underlines and over line) in the N terminus of MEF2D. (B) Effect of mutating QR (10/11) to AA on MEF2D binding to Hsc70. Lysates with over-expressed wt MEF2D or QR (10/11) AA mutant were incubated with GST-Hsc70 in pull-down assays. MEF2D bound to GST-Hsc70 were determined by immunoblotting (n=3, p>0.05).
11 Fig. S6. Effect of α-synuclein on the levels of MEF2D. (A) Inhibition of 6-AN-mediated degradation of MEF2D by α-synuclein. SN4741 cells were transfected with plasmids encoding indicated proteins and treated with 5mM 6-AN for 12 hours. The levels of MEF2D were analyzed by immunoblotting. Graph shows the average values (mean±sem, n=4; * = p<0.05). (B) Inhibition of MEF2D binding to endogenous Hsc70 by α-synuclein. Top panels show the specific interaction between over-expressed MEF2D and endogenous Hsc70 in HEK293 cells. Lysates containing over-expressed MEF2D was immuno-precipitated with anti-mef2d antibody. The precipitates were immuno-blotted for MEF2D (bottom) and Hsc70 (top). Bottom panels show the disruption of MEF2D binding to Hsc70 by α-synuclein. Co-immunoprecipitation with Hsc70 was carried out as described above following over-expression of α-synuclein and MEF2D in HEK293 cells (IB/IP panel: immunoprecipitated and blotted with anti- MEF2D antibody; IB panel: immunoprecipitated with anti-mef2d and blotted with anti- Hsc70 antibody; bottom: immunoblotting of α-synuclein in lysates).
12 Fig. S7. Levels of α-synuclein in brains of Parkinson s disease patients. Photos show representative images of immunohistochemical staining for α-synuclein in the striatum of control (Con) and PD patients (scale bar: 50μm). Graph depicts the relative levels of staining in cases tested (p value is rank sum test).
13 Fig. S8. Impairment of MEF2D binding to DNA following blockade of CMA. (A) Correlation of MEF2D levels and its DNA binding activity. Immunoblotting shows the levels of MEF2D in the lysates used for EMSA in fig. 4A. (B, C) Reduction in MEF2D DNA binding activity following inhibition of CMA. CMA in SN4741 cells was inhibited by either NH 4 Cl (B, cytoplasmic lysates) or anti-sense Hsc70 (C, whole cell lysates). MEF2D DNA binding activity was assessed by EMSA. The bottom immuno blots show the elevated levels of MEF2D following NH 4 Cl or anti-sense Hsc70 treatment. CIAP indicates samples treated with calf intestinal alkaline phosphatase prior to EMSA.
14 Fig. S9. Regulation of MEF2D function by NH 4 Cl. (A) Inhibition of MEF2 transactivation activity by NH 4 Cl. NH 4 Cl inhibits MEF2 transactivation activity measured by MEF2-dependent reporter assay in SN4741 cells (mean±sem, n=5, * = p<0.05). (B) Effect of increasing nuclear MEF2D on NH 4 Cl-induced cell death. Overexpression of MEF2D-VP16 in SN4741 cells attenuates NH 4 Cl-induced loss of viability in WST assay (mean±sem, n=3, * = p<0.05).
15 References 1. D. Zhou et al., Immunity 22, 571 (May, 2005). 2. X. Gong et al., Neuron 38, 33 (Apr 10, 2003). 3. X. Tang et al., J Neurosci 25, 4823 (May 11, 2005). 4. X. Wang, X. Tang, M. Li, J. Marshall, Z. Mao, J Biol Chem 280, (Apr 29, 2005). 5. A. M. Cuervo, E. Knecht, S. R. Terlecky, J. F. Dice, Am J Physiol 269, C1200 (Nov, 1995). 6. A. M. Cuervo, L. Stefanis, R. Fredenburg, P. T. Lansbury, D. Sulzer, Science 305, 1292 (Aug 27, 2004). 7. J. J. Shacka et al., J Neurosci 27, 2081 (Feb 21, 2007). 8. L. J. Martin et al., J Neurosci 26, 41 (Jan 4, 2006).
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