Zdhhc13-dependent Drp1 S-palmitoylation impacts brain bioenergetics, anxiety, coordination and motor skills

Protein S-palmitoylation is a reversible post-translational modification mediated by palmitoyl acyltransferase enzymes, a group of Zn2+-finger DHHC-domain-containing proteins (ZDHHC). Here, for the first time, we show that Zdhhc13 plays a key role in anxiety-related behaviors and motor function, as well as brain bioenergetics, in a mouse model (luc) carrying a spontaneous Zdhhc13 recessive mutation. At 3 m of age, mutant mice displayed increased sensorimotor gating, anxiety, hypoactivity, and decreased motor coordination, compared to littermate controls. Loss of Zdhhc13 in cortex and cerebellum from 3- and 24 m old hetero- and homozygous male mutant mice resulted in lower levels of Drp1 S-palmitoylation accompanied by altered mitochondrial dynamics, increased glycolysis, glutaminolysis and lactic acidosis, and neurotransmitter imbalances. Employing in vivo and in vitro models, we identified that Zdhhc13-dependent Drp1 S-palmitoylation, which acting alone or in concert, enables the normal occurrence of the fission-fusion process. In vitro and in vivo direct Zdhhc13-Drp1 protein interaction was observed, confirming Drp1 as a substrate of Zdhhc13. Abnormal fission-fusion processes result in disrupted mitochondria morphology and distribution affecting not only mitochondrial ATP output but neurotransmission and integrity of synaptic structures in the brain, setting the basis for the behavioral abnormalities described in the Zdhhc13-deficient mice.

body weight, body length, eye, state of whiskers and (Supplementary Fig. S1). The animals (all males) were housed three to four mice/cage and provided with Purina 5058 chow and water ad libitum. The housing room was maintained at 23°C on a 12-h light/dark cycle (lights off at 6 pm). Mice from the three genetic groups appeared in good general health (checked by a thirdparty veterinary staff), without any overt impairments, aberrant responses or unusual levels of activity or fighting during the home cage observation periods. Eight out of nine homozygous and none of eight heterozygous mutant mice showed hair loss in different body areas ( Supplementary Fig. S1). General health observations were performed upon arrival of mice at the behavioral laboratory facility. Behavioral testing began 4-6 days after arrival into the animal facility. Mice were characterized in assays for the presence of motor deficits (open field, rotarod, treadscan, and grip strength), anxiety (open field), and memory (passive avoidance test). Unless otherwise indicated, testing was conducted under fluorescent laboratory lighting. All mice appeared to be healthy at the conclusion of the testing sequence. At this point, when mice were 4-m old, they were euthanized for biochemical analysis.

Behavioral tests
Open field and spontaneous locomotor activity-Spontaneous motor activity and anxietyrelated behavior were recorded with a well-established method [4][5][6] . Over a period of 10  The open field test was conducted in the dark phase. The animal was placed into the center of the box and its behavior was monitored remotely by a videocamera. Measurements were taken of total activity, time spent in certain regions of the grid, number of vertical moves, and stereotypy counts. Animals were monitored for 20-min and then replaced into their home cage. After each animal was tested, the box was cleaned with 10% Nolvasan. This test provides indications of the presence of anxiety or anxiety-like behaviors, as inferred by the center and margin area and time occupied by the animals in the arena. In parallel, routine parameters of general motor behaviors (e.g., rearing, speed, number and length of individual movements) were measured, providing accurate quantification of general locomotor activity 7 .
Grip strength-The grip strength was employed to assess the neuromuscular function as maximal muscle strength of forelimbs and combined forelimbs and hind limbs 8 . Forelimb and hindlimb grip strengths were measured as grasping applied by the mouse on a grid connected to a sensor. Three trials were carried out in succession measuring forelimb-strength only, followed by three successive trials measuring the combined forelimb/hind limb grip strength. The apparatus consisted of a Force gauge meter, with included wire bar grip attachment (model E-DFE-002, Chatillon, Largo, FL) and PC equipped with the Animal Grip Strength System software (model 6910-0002-L, San Diego Instruments, San Diego, CA). For strength measurements, each mouse was lowered over the grid keeping the torso horizontal and allowing only its forepaws to attach to the top of grid before any measurements were taken. Its tail, ensuring the torso to remain horizontal, then gently pulled the mouse back and the maximal grip strength value was recorded.
Three trials were performed for each mouse with a 1-min resting period between trials. In between mice, the wire bar grip was cleaned with 10% Nolvasan.
Treadscan (treadmill)-The use of a treadmill to gather data for comprehensive, quantitative assessment of gait in mice is a sensitive method to evaluate motor performance. The computerassisted footprint analysis assessed gait characteristics such as velocity, stance time, swing time, and stride length as the animal traverses a clear stationary walkway 9,10 . The TreadScan software identified each individual paw of the mouse in each frame as it walked on the treadmill by measuring initial foot contact, stance duration, stride duration, foot liftoff, swing duration, stride length, track width, and toe-spread data for each foot. Gait analysis was evaluated measuring the following outcomes: stance time (paw on the belt), swing time (paw in the air), total stride time, stride length, foot contact area size (also representative of foot pressure), body-foot spacing distance (distances between body and feet), foot spacing distances (distance between various feet), running speed, stride frequencies, foot coupling. The apparatus used consisted of an automated treadmill (Clever Systems, Inc., Reston, VA), a Basler high-speed video camera and PC equipped with BCAM Cap Version 1.00. Each animal was placed into the treadmill enclosure and acclimated for 30 s. The treadmill was then turned on slowly to 20 cm/s and velocity increased to the highest speed tolerated by the animal. Video footage was analyzed using the Treadscan software.
Passive avoidance-The testing apparatus (consisting of Med Associates two chamber box, partition door, shock generator attached to dark chamber, false floor to cover shock grid in light chamber, light centered over light chamber to provide 800 lux brightness, and light shield for dark chamber) was a trough-shaped alley with two distinct compartments separated by a sliding door 11,12 . For the training trial, each mouse was randomly removed from home cage and transferred immediately by tail-hold to the bright chamber. Measurements started as soon as the mouse touched the surface and was released. Ten s after release, the door was opened in the partition and the time taken to enter the second, darkened compartment was recorded. When the animal stepped into the dark section with all four paws, the door was closed and a 2-s footshock was given at 0.5 mA. After 10 s, the cover to the dark chamber was lifted and the mouse was removed and placed in its home cage. The chamber walls floor and shock grid were then cleaned thoroughly with 10% Nolvasan, the partition door removed and the dark chamber cover replaced.
The training phase was repeated the following day. On the third day (time of the testing trial), the animal was returned to the white compartment and the latency to enter the dark compartment (to a maximum of 300 s) was recorded, but no shock was given. The procedure was repeated for all the mice tested.
Acoustic startle-The acoustic startle response was used to assess anxiety levels and sensormotor gating 5 . At the start of each trial, the animal was given 5 min to acclimate to the testing apparatus. Mice were then placed on a platform device that records motion in an enclosed chamber allowing the mice to turn and freely move limbs and tail, but not to rear or ambulate.
Five trial types, 10 trials each for a total of 50 trials per session were run. Trials were randomized within blocks of 5. The trial types were: (i) 120 dB startle alone, (ii) 120 dB startle with 74 dB prepulse, (iii) 120 dB startle with 82 dB prepulse, (iv) 120 dB startle with 90 dB prepulse, and (v) no stimulus -background white noise only. Apparatus frequency was broadband pink noise or white noise surrounding median of 5 khz. Each trial started with a 50 ms null period and ended with a 65 ms recording period. For both periods the background white noise was of 395 (70 dB).

Protein S-palmitoylation quantification
Tissues homogenates in RIPA (1 mg in protein) were pre-cleared of endogenous biotincontaining proteins by immunoprecipitation with streptavidin-coupled dynabeads (Invitrogen) following manufacturer's instructions. Upon removal of the magnetic beads, supernatants were then collected and treated overnight at 4°C with 20 mM N-ethylmaleimide (NEM) to block reduced, non-palmitoylated, cysteine residues. To remove the excess NEM, samples were subsequently precipitate with 10 % PCA for 10 min in ice and centrifugated at 13,000 x g for 10 min. Supernatant was carefully discarded and pellet was resuspended in 200 µl of chilled acetone, followed by centrifugation at 13,000 x g for 10 min. This wash step was repeated for a total of 3 times, excess acetone was eliminated through speed vacuum centrifugation, and the

Isolation of mitochondria from cortex and cerebella of WT, HET and HOM mice
Enriched mitochondrial fractions were obtained from cortex and cerebellum of WT, HET and HOM mice by mechanical cell disruption using a glass-Teflon homogenizer and subsequent centrifugation as previously described 13 . Briefly, after asphyxiation of mice with CO 2 , brains were quickly removed and cerebella and cortex were washed in cold 0.25 M sucrose, blotted and weighed. Then, they were placed in 0.22 M mannitol, 70 mM sucrose, 0.5 mM EGTA, 2 mM HEPES, 0.1% fatty acid-free BSA, pH 7.4 (MSHE), and homogenized in glass-Teflon homogenizer using a 5:1 buffer to brain wet weight ratio. Large cell debris and nuclei were pelleted by centrifuging at 600 × g for 5 min. Mitochondria were subsequently pelleted by centrifuging the supernatant for 10 min at 10,300 × g. This mitochondrial fraction was tested for enrichment and purity by performing proteomics of mitochondrial and post-mitochondrial fractions.

Cell culture conditions
Neuronal progenitor cells (NPC) were obtained from the CHDI repository program and grown as previously described in detail 14 . After thawing cells were plated in T75 flasks and grown at 33°C in a humidified atmosphere containing 5% CO 2 with 20 ml of Dulbecco's Modified Eagle Medium supplemented with 10% FBS (Hyclone #SH30071.03) and 10 4 IU/ml penicillin and 10 4 µg/ml streptomycin (Gibco). When at 80-90% confluence, the cells were plated in T75 flasks at a cell density of 2.5 × 10 5 per flask and selected in DMEM with 400 µg/ml G418 (Invitrogen). Cells were grown at 33°C for 3-5 days, changing the media every 2 to 3 days, until 80-90% confluent and then harvested by trypsinization, counted with a TC-10 cell counter (Bio-Rad) with 0.1% Trypan Blue.

Evaluation of mitochondrial distribution and morphology by confocal microscopy in cells in culture
Striatal progenitor cells (1 × 10 5 ) were seeded on sterile coverslips, grown overnight at 33°C, Immunoreactivity was visualized using an Alexa-Fluor555-conjugated anti-rabbit IgG (Invitrogen #A32732) secondary antibody for 1 h at room temperature. DNA was stained with DAPI. Fluorescent images were obtained using an Olympus FV1000 laser scanning confocal microscope with either a 40X or a 60X objective. Mitochondrial morphology was evaluated by ImageJ using the parameters described in detail before 14 . Estimates of the proportion of mitochondria recovered and the degree of purification achieved in each of the fractions named "cytosol" and "mitochondria (n = 7 mice) was obtained using proteomics analysis in each of these fractions. Data are shown as the average of 7 animals. In assessing the suitability of mitochondrial preparation for our studies, the degree of contamination  Calcium/calmodulin-dependent serine/threonine kinase involved in cell survival, apoptosis, and autophagy.

Q8K1M6 Dynamin-1-like protein Dnm1l
Mediates mitochondrial membrane fission through a GTP hydrolysis-dependent mechanism. Required for normal brain development, including that of cerebellum.

P16858
Glyceraldehyde-3phosphate dehydrogenase Gapdh Key enzyme in glycolysis. Modulates the organization and assembly of the cytoskeleton. Also participates in transcription, RNA transport, DNA replication and apoptosis.

P62827
GTP-binding nuclear protein Ran

Ran
GTPase involved in nucleo-cytoplasmic transport of proteins and RNAs. Controls cargo loading and release by transport receptors in the proper compartment and ensure the directionality of the transport.

Q80X52
Mitochondrial glutamate carrier 1 Slc25a22 Involved in the transport of glutamate across the inner mitochondrial membrane.

Syntaxin-1B Stx1b
Potentially involved in docking of synaptic vesicles at presynaptic active zones.

Unconventional myosin-Va Myo5a
Actin-based motor. Mediates the transport of vesicles to the plasma membrane. May also be required for some polarization process involved in dendrite formation.
Drp1 or Zdhhc13 were pulled down by IP of brain lysates from WT or KO mice. The resulting pellets under each condition were analyzed by mass spectrometry. Quantification was performed by spectral counting. Abundance of proteins pulled down in WT were subtracted from those pulled down in KO mice (used as background) for each antibody used. Once these two lists of proteins were obtained (one with each antibody), only common proteins were selected. Mass spectrometry was performed at the UCD Facility.

Legends to Supplementary Figures and Dataset
Supplementary Figure  All mice utilized in this study were genotyped for the wild-type or truncated Nnt as described 16,17 . Genomic DNA was extracted from tails using a Qiagen DNeasy tissue kit. Concentration and purity of DNA was measured at an absorbance of 260 nm and 280 nm on a Tecan Infinite M200 Nanoquant (Tecan, Austria). The Nnt allele expressed by the B6/NJ substrain was PCR genotyped using a three primer, two allele PCR assay that discriminates between the Nnt wild-