TITLE: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy
AUTHORS: Capucine de Talhouet, Benjamin O'Callaghan, Helene Plun-Favreau, Noemi Esteras, Marc P. M. Soutar

[DESCRIPTION]
It has recently been shown that KAT8, a genome-wide association study candidate risk gene for Parkinson’s Disease, is involved in PINK1/Parkin-dependant mitophagy. The KAT8 gene encodes a lysine acetyltransferase and represents the catalytically active subunit of the non-specific lethal epigenetic remodelling complex. In the current study, we show that contrary to KAT5 inhibition, dual inhibition of KAT5 and KAT8 via the MG149 compound inhibits the initial steps of the PINK1-dependant mitophagy process. More specifically, our study shows that following mitochondrial depolarisation induced by mitochondrial toxins, MG149 treatment inhibits PINK1-dependant mitophagy initiation by impairing PINK1 activation, and subsequent phosphorylation of Parkin and ubiquitin. While this inhibitory effect of MG149 on PINK1-activation is potent, MG149 treatment in the absence of mitochondrial toxins is sufficient to depolarise the mitochondrial membrane, recruit PINK1 and promote partial downstream recruitment of the autophagy receptor p62, leading to an increase in mitochondrial delivery to the lysosomes. Altogether, our study provides additional support for KAT8 as a regulator of mitophagy and autophagy processes.



[STEPS]
SECTION: Cell Culture
1. Maintain cells in culture in a humified 5% CO2 incubator at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Gibco, 11995-065) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco). Change cell medium every 3 days. 
 
 

SECTION: Cell Culture
2. Split cells at 75% to 90% confluency using 2 mL of Trypsin.

SECTION: Cell Treatment (Small Molecule Modulator and Mitophagy Induction)
3. Dilute each compound in DMSO to reach concentrations determined according to their speculated toxicity and IC50, 10 micromolar (µM) for NU9056 and 100 micromolar (µM) for MG149.

 

SECTION: Mitochondrial Enrichment
7.   Thaw, scrape into 1.5mL centrifuged tubes, and triturate 20x, keeping samples on ice .
SECTION: Mitochondrial Enrichment
8.    Freeze tubes at -80 °C for 60 min , then thaw on ice .
SECTION: Mitochondrial Enrichment
9. Remove cell debris / non-lysed cells by centrifugation 1500 rcf, 20 min, 4 °C .

SECTION: Mitochondrial Enrichment
12. Determine the relative ratios of sample differences for the mitochondrial enriched fraction using the protein concentration of the cytoplasmic fraction (BSA assay). Re-suspend the mitochondrial enriched pellet accordingly using  1x NuPAGE® LDS sample buffer supplemented with 10 mM dithiothreitol (DTT).
SECTION: Cell Lysis
14. Lyse cells in whole-cell lysis buffer (50mM Tris (pH 7.4), 0.1mM EGTA, 1mM EDTA, 0.27M Sucrose, 1% triton-x-100, supplemented with protease and phosphatase inhibitors)on iceand incubated    at -80 °C. 
SECTION: Mitochondrial Enrichment
11. Centrifuge the cytoplasmic fraction 12500 x g, 20 min, 4 °C to remove membrane contamination and wash the mitochondrial fraction twice in 500 µL of fractionation buffer (12500 x g, 10 min, 4 °Cx 2) to remove cytoplasmic contaminants.
SECTION: Mitochondrial Enrichment
10. Centrifuge the supernatant 12500 x g, 20 min, 4 °C to collect the mitochondrial enriched pellet (aka the mitochondrial fraction) and save the supernatant (aka the cytoplasmic fraction).
SECTION: Mitochondrial Enrichment
13.   Sonicate the mitochondrial fraction (10 microns power amplitude for 2 x 10 seconds, "flick" in 
  between) and heat to 70 °C  for 10 min.  
SECTION: Cell Lysis
15. Centrifuge the lysates 1600 x g, 20 min, 4 °C and protein quantify the supernatant by BSA assay. 
SECTION: Western Blotting
17. Separate the LDS preps (either after cell lysis or mitochondrial enrichment) by SDS-PAGE and transfer them to nitrocellulose membrane.
SECTION: Western Blotting
18. Incubate the membrane   at 4 °C with primary antibodies diluted in 3% milk in PBS-Tween.
SECTION: Western Blotting
19. Wash and incubate the membrane for 60 min at Room temperature in secondary antibodies diluted in PBS-Tween.

SECTION: Western Blotting
20. Detect the protein bands using Odyssey CLx LI-COR.
SECTION: Immunofluorescence
21. Fix cells with 4% Paraformaldehyde (PFA, Sigma-Aldrich) for 20 min atRoom temperature. 
SECTION: Immunofluorescence
22. Remove PFA solution, block and permeabilize the cells with a mix of 10% FBS, 0.25% Triton X-100 in PBS for 60 min at Room temperature .
SECTION: Immunofluorescence
23. Immunostain the cells with pUb(Ser65) and TOM20 primary antibodies diluted in 10% FBS/PBS for 120 min at Room temperature.

 

SECTION: Immunofluorescence
24. Wash wells three times using PBS 1X and incubate with secondary antibodies for 60 min at Room temperature , before washing again three times using PBS 1X. 
 

SECTION: Immunofluorescence
25. Image cells on the Opera Phenix (Perkin Elmer), acquiring confocal z-stacks for multiple fields of view across 3 individual wekks per experimental condition, using the 40X water objective. 
 

SECTION: Measuring Mitochondrial Membrane Potential using TMRM
26. Seed POE-SHSY4Y cells in a 96-well CellCarrier Ultra plate.
SECTION: Immunofluorescence
26. For visualisation purposes, select brightness and contrast settings on the control and apply the same settings to all other conditions.
26. 
SECTION: Mitochondrial Membrane Potential Measurement using TMRM
27. Incubate live cells in 25 nanomolar (nM)TMRM Perform Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) measurements in redistribution mode where a decrease in TMRM signal intensity is associated with mitochondrial membrane potential (ψm) depolarisation (confirmed post-imaging by treatment with 1 micromolar (µM) Carbonyl cyanide m-chlorophenyl hydrazone, CCCP, ψm depolarising agent).




Incubate live cells in 25 nanomolar (nM) TMRM (Sigma Aldrich) diluted in Hanks' Balanced Salt Solution (HBSS, Gibco) buffered with 10 millimolar (mM) HEPES (Sigma Aldrich), 7.4 for 00:40:00 at 37 °C. 




68Image cells on the Opera Phenix (PerkinElmer) at 37 °C, acquiring confocal z-stacks for 27 fields of view across 3 individual wells per experimental condition using the 40X water objective, NA1.1. 
Note
Excite TMRM with the 561 nm laser (50% power) and collect signal with 570-630nm emission filter.
Excite TMRM with the 561 nm laser (50% power) and collect signal with 570-630nm emission filter. 






69Analyse images in an automated way using the Columbus 2.8 analysis system (Perkin Elmer) to measure the TMRM signal intensity of maximum intensity projections.
SECTION: Statistical Analysis
26. Normalise intensity measurements from imaging experiments for each independent, replicate experiment. Calculate the mean value from a minimum of 3 technical replicates for each condition of an independent, replicate experiment.
SECTION: Statistical Analysis
29. Subject the data to a two-way ANOVA with Dunnett's post-hoc analysis for multiple comparisons, unless otherwise stated

 

SECTION: Statistical Analysis
28. Normalise integrated density measurements from Western blot experiments to control conditions.
SECTION: Cell Lysis
16. Make LDS preps with 10 mM dithiothreitol (DTT), and heat to 70 °C  for 10 min. 
SECTION: Measuring Mitochondrial Delivery to Lysosomes using mitoKeima Reporter
29. Seed mt-Keima expressing POE SHSY5Y cells in a 96-well CellCarrier Ultra plate and treat them according to desired conditions (MG149, NU9056, O/A as described in  ).
SECTION: Measuring Mitochondrial Membrane Potential using TMRM
28. Image cells on the Opera Phenix (PerkinElmer) at 37 °C and 4% CO2, acquiring confocal z-stacks for multiple fields of view across 3 individual wells per experimental condition using the 40X water objective. 

 

SECTION: Measuring Mitochondrial Membrane Potential using TMRM
27. Incubate live cells in25 nanomolar (nM) Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) (Sigma Aldrich) diluted in cell media for 40 min at 37 °C and 4% CO2.
SECTION: Measuring Mitochondrial Delivery to Lysosomes using mitoKeima Reporter
30. Before imaging, replace cell media with phenol-free DMEM + 10% FBS containing the treatments previously administered  , and adding Hoechst 33342 (1:10000) to stain the nuclei.
SECTION: Measuring Mitochondrial Delivery to Lysosomes using mitoKeima Reporter
31. Image cells immediately on the Opera Phenix (PerkinElmer) at 37 °C with 5% CO2, acquiring multiple single-plane fields of view, using the 63X water objective. 

 

SECTION: Mitochondrial Enrichment
6. Incubate cells    at -80 °C in mitochondrial homogenization buffer (250mM sucrose, 1mM EDTA, 10mM Tris (pH 7.4) supplemented with protease and phosphatase inhibitors).
SECTION: Cell Treatment (Small Molecule Modulator and Mitophagy Induction)
5. Treat cells with either DMSO or 10 micromolar (µM) oligomycin/ 10 micromolar (µM) antimycin for 180 min to induce mitophagy before collecting / fixing for downstream experiments. 

 


SECTION: Cell Treatment (Small Molecule Modulator and Mitophagy Induction)
4. Treat cells with DMSO,  10 micromolar (µM) NU9056, or 100 micromolar (µM) MG149 for 360 min before collecting / fixing for downstream experiments.

SECTION: RT-qPCR
32. Extract total RNA from cells using the Monarch Total RNA Miniprep Kit (New England Bioscience) with inclusion of the optional on-column DNAse treatment.
SECTION: RT-qPCR
33. Reverse transcribe RNA with SuperScript™ IV reverse transcriptase and random hexamers (Invitrogen).
SECTION: RT-qPCR
34. Dilute the cDNA product then subject to quantitative real-time PCR (qPCR) using Power SYBR™ Green Master Mix (Applied Biosystems) and gene specific primer pairs on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems). 

     ABCD1       Sequence 5'-3'       2  Gene Target    Forward    Reverse    Product Size / bp  3  RPL18A    CCCACAACATGTACCGGGAA    TCTTGGAGTCGTGGAACTGC    180  4  UBC    CTGGAAGATGGTCGTACCCTG    GGTCTTGCCAGTGAGTGTCT    117  5  KANSL1    ATCCTCCACACAGTCCCTTG    CCCCTTCTCCTCCTTACTGG    121  6  KAT8    TCACTCGCAACCAAAAGCG    GATCGCCTCATGCTCCTTCT    107  7  PINK1    GTGGAACATCTCGGCAGGTT    CCTCTCTTGGATTTTCTGTAAGTGAC    129  8  PRKN    ACAAATAGTCGGAACATCACTTGCA    AACAAACTGCCGATCATTGAGTCTT    144  9  MAPT    TGCAAATAGTCTACAAACCAGT    CTTGTGGGTTTCAATCTTTT    209  

SECTION: RT-qPCR
35. Calculate relative mRNA expression levels using the 2−ΔΔCt method and RPL18A as the housekeeping gene.

 
SECTION: Statistical Analysis
36. Use a minimum of 3 biological replicates for each experiments (N numbers shown in legends refer to the number of replicate experiments).
- For each imaging experiments, calculate the mean values of every condition from a minimum of 3 technical replicates.
- Normalise intensity and integrated density measurements to control conditions.
SECTION: Statistical Analysis
37. Subject data to a two-way ANOVA with Dunnett's post-hoc analysis for multiple comparisons unless stated otherwise.
- Express data as the mean ± standard deviation (SD) from replicate experiments.
- Set statistical significance at a p-value of <0.05. 

 

SECTION: Measuring p62 recruitment to the OMM and mitochondrial delivery to lysosomes using mitoSRAI
38. Seed mitoSRAI POE SHSY5Y cells in a 96-well CellCarrier Ultra plate and treat them according to desired conditions (MG149, NU9056, O/A as described in ).

SECTION: Measuring p62 recruitment to the OMM and mitochondrial delivery to lysosomes using mitoSRAI
39. Fix cells as described in   

SECTION: Measuring p62 recruitment to the OMM and mitochondrial delivery to lysosomes using mitoSRAI
40. Immunostain the cells with p62 primary antibodies diluted in 10% FBS/PBS for 120 min at Room temperature.

 

SECTION: Measuring p62 recruitment to the OMM and mitochondrial delivery to lysosomes using mitoSRAI
41. Wash wells three times using PBS 1X and incubate with secondary antibodies for 60 min at Room temperature , before washing again three times using PBS 1X. 
 

SECTION: Measuring p62 recruitment to the OMM and mitochondrial delivery to lysosomes using mitoSRAI
42. Image cells on the Opera Phenix (Perkin Elmer), acquiring confocal z-stacks for multiple fields of view across 3 individual wells per experimental condition, using the 63X water objective. 
 

SECTION: Generating PINK1 KO POE SH-SY5Y cell line
43. PINK1 was functionally knocked out from the POE SH-SY5Y cell line through CRISPR-Cas9 editing by adapting previously published protocols (DOI: 10.15252/emmm.202013579)

 

 

SECTION: Generating PINK1 KO POE SH-SY5Y cell line
44. A subconfluent 6-well dish of POE SH-SY5Ys were transfected with 1ug of each PINK1_Seq1 and PINK1_Seq2 sgRNA encoding pSpCas9(BB)-2A-GFP plasmid using lipofectamine 3000 (Invitrogen)
SECTION: Generating PINK1 KO POE SH-SY5Y cell line
45. 24 h post-transfection cells were trypsinised and subjected to fluorescence-activated cell sorting and clonal selection of GFP-positive cells: isolated clones were expanded and phenotypically screened for clones which did not show pUb(Ser65) signal after 1.5 h O/A treatment
SECTION: Generating mitoSRAI POE SH-SY5Y cell line
48. MitoSRAI cDNA was first PCR amplified from mitoSRAI_pcDNA3 with inclusion of 5′ EcoRI and 3′ NotI restriction sites

SECTION: Generating mitoSRAI POE SH-SY5Y cell line
49. The PCR product was cloned into pLVX-EF1α-IRES-Puro using EcoRI and NotI restriction sites to generate pLVX-EF1α-mitoSRAI-IRES-Puro. 
 

SECTION: Generating mitoSRAI POE SH-SY5Y cell line
50. Lenti-X 293 T HEK cells cultured in DMEM 10% FBS media were transfected with pMD2.G, pCMVR8.74 and pLVX-EF1α-mitoSRAI-IRES-Puro at a 1:1:2 molar mass ratio using Lipofectamine 3000 (Invitrogen)
SECTION: Generating mitoSRAI POE SH-SY5Y cell line
51. The next day, a full media change was performed using DMEM 10% FBS and cells cultured for further 24 
SECTION: Generating mitoSRAI POE SH-SY5Y cell line
52. The lentivirus containing media was collected and diluted 1:2 with DMEM 10% FBS before filtering through 0.44 µm PES filters
SECTION: Generating mitoSRAI POE SH-SY5Y cell line
53. 1 × 106 POE SHSY5Ys were reverse transduced with the 1:2 lentivirus supernatant dilution in the presence of 10 µg/mL polybrene (Sigma Aldrich)
SECTION: Generating mitoSRAI POE SH-SY5Y cell line
54. Bulk populations of cells stably expressing mitoSRAI were established through selection with 1 ug/mL puromycin (M P BIOMEDICALS UK)
Puromycin was maintained during routine culture but withdrawn when seeding for experimental assays.
SECTION: Generating PINK1 KO POE SH-SY5Y cell line
47. gDNA was extracted from a positive clone using the Wizard Genomic DNA Purification Kit (Promega) and subjected to whole genome sequencing (Novogene) which revealed homozygous 22 bp deletion and frameshift in exon 1 of PINK1 (c.156_177delGGGCGCGGAGCCTCGCAGGGTC, p.A54Sfs*46).
SECTION: Generating PINK1 KO POE SH-SY5Y cell line
46. Confluent wells of a 96-well were prepared for immunofluorescence staining of pUb(Ser65) with IRDye 800CW donkey anti-rabbit secondary