PMC

In primary cortical neurons, P259 inhibits basal physiological fission, resulting in mitochondrial dysfunction and loss in motility. (a) Top, immunofluorescence images depicting mitochondrial morphology (Tom20) of E17 primary cortical neurons after a 2-hour treatment with 1 µM TAT, 1 µM P259, or 10 µM Mdivi-1 (n = 3, 10 images each). Bottom, quantification of the form factor (degree of branching) and aspect ratio (major/minor axis length). At least 10,000 mitochondrial fragments were analyzed for each condition. Mitochondrial reactive oxygen species production (MitoSox Red) (b) and membrane potential (TMRM) (c) after overnight treatment with indicated concentrations of peptide (n = 3, with 3 replicates). (d) E17 primary cortical neurons treated with 4 µM P259 or no peptide control and stained with 50 nM TMRM. Overlay of live microscopy imaging at time 0 (green) and 150 (red) seconds. Bottom left: the corresponding bivariate kernel density estimate plots of the 0 sec versus 150 sec TMRM signal at each pixel for the represented images (n = 4 control and 6 P259, 3 movies each). An estimation of the % mobile mitochondria from the Pearson coefficient of each set of images. Data are mean ± s.e.

Rational peptide design identifies a novel inhibitor of the Mff-Drp1 protein-protein interaction. (a) A scheme of the rationale behind the inhibitor design. (b) A short sequence of homology between Mff and Drp1 representing P259 identified by lalign. ‘:’ and ‘.’ correspond to complete identity and sequence similarity, respectively. (c) P259 (purple) is derived from the GTPase domain of Drp1. Surface and ribbon diagrams of Drp1 structure (PDB, 4BEJ) with the GTPase, bundle signaling element (BSE), and stalk domains highlighted. (d) A heatmap representing sequence conservation of human proteins that contain a region similar to P259. Sequence conservation corresponds to the number of residues similar/identical to P259 from each sequence. (e) Binding of 100 nM recombinant human Drp1, Mff, and Mfn2 to 50 nM surface-conjugated P259. The obtained graph is of the mean standardized response and ± s.e. as a function of time for n = 3 individual experiments, with 3 technical replicates each. (f) K_D values for each protein-peptide interaction at 100 nM protein concentration (n = 3, with 3 replicates) (g) Co-immunoprecipitation of recombinant Drp1 (200 ng) with each adaptor (200 ng) in the presence/absence of 1 µM P259 (a representative blot of two independent experiments). Represented Western blot is cropped from a full blot shown in the Expanded Western Blots section of the Supplementary Information. (h) Relative GTPase activity of recombinant Drp1 in the presence of 1 µM of peptide (n = 3–5). Data are mean ± s.e. from 3–5 independent experiments per condition.

P259 inhibits the Mff/Drp1 protein-protein interaction and physiological mitochondrial fission in SH-SY5Y cells, resulting in mitochondrial dysfunction. (a) Co-immunoprecipiation of Drp1 and Mff from SH-SY5Y cell lysate treated with 1 µM P259 for 1 hour (n = 3, with 2 replicates). Represented Western blot is cropped from a full blot shown in the Expanded Western Blots section of the Supplementary Information. (b) Immunofluorescence images depicting mitochondrial morphology after a 30-minute treatment with 4 µM TAT, 4 µM TAT-P259 (P259), or 10 µM Mdivi-1. Hoechst 33342 is shown in blue and Tom20 in green (n = 3, 10 images each). Below, quantification of the form factor (degree of branching) and major axis length (length of mitochondria). At least 6,000 mitochondrial fragments were measured for each condition. Mitochondrial reactive oxygen species (ROS) production (MitoSox Red) (c) and mitochondrial membrane potential (TMRM) (d) after overnight treatment with indicated concentrations of peptide or FCCP. Data are from 3 independent experiments per condition normalized to the average no peptide value. (e) Relative mitochondrial ROS (MitoSox Red) signal in GFP positive cells expressing full-length or alanine-substituted P259 at indicated amino acids after two days of transfection. Values are normalized to a GFP empty vector (n = 3, with 3 replicates). (f) The effect of overnight treatment with 4 µM TAT or P259 on electron transport chain flux as measured through the oxygen consumption rate (OCR) (n = 3, with at least 3 replicates). Data are mean ± s.e.

P259 inhibits physiological mitochondrial fission, lowers brain ATP levels and causes behavioral deficit in wild-type mice, and accelerates progression of disease in an R6/2 Huntington’s mouse model. WT and R6/2 mice were treated with 3 mg/Kg/day of P259 or TAT for 8 weeks starting at week 6 of age. (a) Quantification of mitochondrial Drp1 content relative to ALDH2 (a mitochondrial matrix enzyme) in the mitochondrial fraction isolated from total brain lysate of wild-type mice (Western blot shown in Fig. S) (n = 4 mice). (b) ATP content in brain tissue of wild-type mice (n = 4 mice, 2 replicates). (c) Representative transmission electron microscopy images of the striatum from wild-type mice. Quantification of mitochondrial area of 806 (TAT) and 1109 (P259) mitochondria from ten images per condition. (d) Active mouse behaviors (sum of time rearing, jumping and digging, in seconds), total distance moved (cm), jump counts, and combined jumping and climbing duration (sec) following 8 weeks of treatment with P259 (n = 10 mice). (e) Principal component analysis of the top 20% of behaviors collected from wild-type mice treated with 3 mg/Kg/day TAT (black)/P259 (purple) for 12 weeks. The first principal component represents 42% of the variation. (f) Local linear embedding dimensionality reduction and visualization of all behaviors collected weekly from TAT- (black) or P259- (purple) treated WT and TAT- (white) treated R6/2 mice over the course of 8 weeks. (g) Survival of WT and R6/2 mice treated with either TAT or P259 continuously at 3 mg/mL. All behavior and survival tests were conducted by an experienced observer who was blinded to the treatment groups. Data are mean ± s.e.