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. 2017 Oct 16;7(1):12796.
doi: 10.1038/s41598-017-12889-0.

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

Eleonora Napoli  1 , Gyu Song  1 , Siming Liu  1 , Alexsandra Espejo  2 , Carlos J Perez  2 , Fernando Benavides  2 , Cecilia Giulivi  3   4
Affiliations

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

Eleonora Napoli et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Skin phenotype, tissue gene expression and S-palmitoylation levels in Zdhhc13 deficiency. (a) Hair coat characteristics of luc mice. Pictures taken by Dr. F. Benavides. (b) Zdhhc13 gene expression was performed by qPCR in skin, brain and lung from WT and luc mice. Statistical analyses were performed with Student’s t test. (c) S-palmitoylation detection in cytosolic and mitochondrial-membranes fractions from cerebellum of WT and HOM mice. Bars indicate proteins with decreased palmitoylation levels in HOM vs. WT. Molecular weights (in kD) were calculated with the Carestream software based on the MagicMark XP Western Protein Standard. Further details are reported in the Methods section. HA = hydroxylamine.
Figure 2
Figure 2
Evaluation of locomotor activity, coordination, gait and anxiety in luc mice. Representative outcomes are shown for 3-m old WT (n = 8), HET (n = 8) and HOM (n = 9; see Methods for details). All outcomes data are shown as mean ± SEM for each genotype and statistical analysis was performed by ANOVA followed by Bonferroni’s post-hoc test, unless otherwise noted. P values are reported right below each test. Other activities and locomotion parameters recorded are shown in Table 1 and Supplementary Fig. S2. P values are as follows. Open field: <0.0001 for a,b,g; 0.0021; 0.007 for d,i; 0.03 for e,j; 0.045 for f; 0.029 for h. Rotarod: Statistical analysis was performed with a 2-way ANOVA, using time and genotype as factors for the analysis, followed by Bonferroni’s post-test for multiple comparisons. P values are as follows: 0.0039 for a,b; 0.017 for c; 0.0009 for d; 0.0076 for e; 0.0005 for f; 0.0404 for g; 0.0064 for h; 0.013 for i; 0.030 for j; 0.0027 for k; 0.018 for l and o; 0.0022 for m; 0.021 for n. Acoustic startle response: *p = 0.014 **p = 0.0085 vs WT. Treadmill: Reported are outputs significantly different among the three groups. P values are as follows: 0.049 for a,b; 0.0063 for c; 0.018 for d; 0.036 for e; 0.045 for f; 0.0037 for g; 0.0302 for h; 0.0037 for i,j; 0.044 for k; 0.028 for l; 0.0082 for m; 0.019 for n; 0.0058 for o and p; 0.028 for q; 0.0097 for r; 0.0002 for s.
Figure 3
Figure 3
Effect of age and Zdhhc13 deficiency on brain mitochondrial outcomes. Cerebellar and cortical activities of Complex I, IV and V, expressed as nmol x (min x mg protein)−1, were normalized by citrate synthase activity. These outcomes were obtained from 3-m old WT (n = 3), HET (n = 8), HOM (n = 8) mice after the behavioral testing was performed, or from 2-y old WT (n = 12), HET (n = 6), HOM (n = 6). Statistical analysis was performed via 2-way ANOVA to evaluate the effect of genotype, time and their interaction on the total variance. In bold are statistically significant effects, and between parentheses are values of F (DFn, DFd). Bonferroni’s post-hoc test for multiple comparisons was also performed and statistically significant p values are as follows: a, b, e, f, g, h = <0.0001; c = 0.0102; d = 0.0014; i = 0.0003; j = 0.0002. All other details were included in the Methods section and Supplementary Methods.
Figure 4
Figure 4
Untargeted metabolomics of cerebella from WT, HET and HOM mice. This analysis was performed in cerebella from 3-m old WT (n = 3), HET (n = 6) and HOM (n = 6) luc mice. Levels of metabolites statistically significantly different in at least one of the paired comparisons (WT-HET and/or WT-HOM) are shown. Lower panel: Pathway over-representation analysis was run with MetaboAnalyst. The size of the symbols is proportional to the impact. In dark gray, pathways with FDR < 0.05. The rest are indicated in light gray. The numbers were used to identify each of the pathways.
Figure 5
Figure 5
Scheme representing differences in metabolic flow of substrates in the presence of Zdhhc13 loss-of-function. Metabolites with differential levels in cerebella from WT and luc mice (from Fig. 4) are shown in light gray (higher abundance in luc than WT) and in italics (lower levels in luc than WT). Gray arrows indicate lower flux whereas black, enhanced pathways.
Figure 6
Figure 6
Evidence of the direct protein interaction between Zdhhc13 and Drp1. (a) Representative cropped image of Drp1, VDAC and actin levels in cytosolic and mitochondrial cerebellar fractions from WT, HET and HOM mice (n = 3). Data are reported as mean ± SEM. Statistical analysis was performed by ANOVA, followed by Bonferroni’s post-hoc test. P values are as follows: a = 0.015; b = 0.049. (b) Mitochondria (red) morphology, distribution and mass, and Drp1 levels (green) in NPC vehicle-treated (a,b) and treated (c,d) with 20 µM 2-bromo-palmitate for 6 h. Pictures were taken at a 120x magnification. Mitochondrial Drp1 levels were evaluated using the Image J (Fiji) colocalization plugin and reported as mean ± SEM of ≥10 fields. Statistical analysis was performed with paired Student’s t test. (c) Drp1, beta-subunit of mitochondrial ATPase and actin levels in total and mitochondrial fractions of NPC before and after treatment with 20 µM 2-bromo-palmitate for 6 h. Densitometry was evaluated with the Carestream software and data are reported as mean ± SEM of 2 separate experiments. Statistical analysis performed with paired Student’s t test. (d) Complex IV and V activities are shown as either normalized or non-normalized by citrate synthase. Specific activities are expressed as nmol x (min × 106 cells)−1. Statistical analysis performed by paired Student’s t test between vehicle-treated and bromo-palmitate-treated samples. (e) Cerebellar Drp1 palmitoylation levels (cropped images). Immunoprecipitation of Drp1 was followed by ABE assay (see Methods). Levels of S-palmitoylated Drp1 normalized to total Drp1 are reported. HA = hydroxylamine. (f) Interaction of purified, recombinant Drp1 with full length and truncated GST-tagged Zdhhc13. The kinetics traces were obtained with WT and mutant Zdhhc13 at concentrations indicated in the text. Other details, see Methods. (g) Representative cropped image showing Drp1 levels in WT and HOM brains upon immunoprecipitation with anti- Drp1 or Zdhhc13 antibodies. Densitometry analysis was performed with Image J. The antibody heavy chain was used as loading control. Statistical analysis was performed with the Student’s t test. Representative full-length western blot images are shown in Supplementary Fig. S5.
Figure 7
Figure 7
Graphical abstract of the role of Zdhhc13 in mitochondria dynamics. In luc mice or through pharmacological inhibition of S-palmitoylation, lower S-palmitoylation of Drp1 (Palm-Drp1) leads to impaired translocation to mitochondria. This results in an imbalance between the fusion and fission processes, interfering with mitophagy and/or segregation of mitochondria into daughter cells. Accumulation of defective mitochondria results in lower ATP production, and in the case of neurons, imbalances in neurotransmission. Both processes are likely to result in behavioral deficits observed in the luc mice.
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