Alterations of lipid‐mediated mitophagy result in aging‐dependent sensorimotor defects

Abstract The metabolic consequences of mitophagy alterations due to age‐related stress in healthy aging brains versus neurodegeneration remain unknown. Here, we demonstrate that ceramide synthase 1 (CerS1) is transported to the outer mitochondrial membrane by the p17/PERMIT transporter that recognizes mislocalized mitochondrial ribosomes (mitoribosomes) via 39‐FLRN‐42 residues, inducing ceramide‐mediated mitophagy. P17/PERMIT‐CerS1‐mediated mitophagy attenuated the argininosuccinate/fumarate/malate axis and induced d‐glucose and fructose accumulation in neurons in culture and brain tissues (primarily in the cerebellum) of wild‐type mice in vivo. These metabolic changes in response to sodium‐selenite were nullified in the cerebellum of CerS1to/to (catalytically inactive for C18‐ceramide production CerS1 mutant), PARKIN−/− or p17/PERMIT−/− mice that have dysfunctional mitophagy. Whereas sodium selenite induced mitophagy in the cerebellum and improved motor‐neuron deficits in aged wild‐type mice, exogenous fumarate or malate prevented mitophagy. Attenuating ceramide‐mediated mitophagy enhanced damaged mitochondria accumulation and age‐dependent sensorimotor abnormalities in p17/PERMIT−/− mice. Reinstituting mitophagy using a ceramide analog drug with selenium conjugate, LCL768, restored mitophagy and reduced malate/fumarate metabolism, improving sensorimotor deficits in old p17/PERMIT−/− mice. Thus, these data describe the metabolic consequences of alterations to p17/PERMIT/ceramide‐mediated mitophagy associated with the loss of mitochondrial quality control in neurons and provide therapeutic options to overcome age‐dependent sensorimotor deficits and related disorders like amyotrophic lateral sclerosis (ALS).

Cer levels (left) and 17C-ceramides species synthesized in vitro by the mitochondrial and microsomal fractions of the brain tissue freshly isolated from CerS1 to/to and WT mice treated with 1 mg/kg of SoSe for 3 hours.Measurements of ceramides were normalized to total inorganic phosphate in the probe.b, Western blot of CerS1 levels in the whole lysate (WL, input) and in microsomal and mitochondrial fractions of the brain tissues isolated from animals from a. Actin was used as a loading control.TOM20 and Calnexin as loading controls for mitochondrial and microsomal fractions, respectively.c, Mass-spectrometry analysis of 17CdhC18-Cer levels (left) and 17Cdh-ceramides species synthesized in vitro by the mitochondrial and microsomal fractions of the brain tissue freshly isolated from CerS1 to/to and WT mice treated with vehicle or 1 mg/kg of SoSe for 3 hours.Measurements of ceramides were normalized to total inorganic phosphate in the probe.d, Western blot of CerS1 levels in the whole lysate (WL, input).Actin was used as a loading control.e, Levels of CerS1 in microsomal and mitochondrial fractions of the brain tissues isolated from animals from c. TOM20 and Calnexin as loading controls for mitochondrial and microsomal fractions, respectively.f, Mass-spectrometry analysis of 17CdhC18-Cer levels (left) and 17Cdhceramides species (right) synthesized in vitro by the mitochondrial and microsomal fractions of Scr control and shICT1 1A cells and treated with vehicle or SoSe (10 µM for 3 hours).
Measurements of ceramides were normalized to total inorganic phosphate in the probe.g, Western blot of CerS1 levels in the whole lysate (WL, input).Actin was used as a loading control.h, CerS1 levels in microsomal and mitochondrial fractions of the 1A cells from g. TOM20 and Calnexin as loading controls for mitochondrial and microsomal fractions, respectively.i, Levels of ICT1 in Scr   The pump was assembled, loaded with LCL768 (1 mg/kg; 0.25 ml/h for 28 days), and equilibrated for 48 h at 37 oC.This was followed by intracranial implantation as described by the manufacturer.
Behavioral studies in mice.Accelerated Rotarod test: The mice were placed in the rotating cylinder twice daily for three days.Each trial lasts a maximum of 10 min, during which the rotating rod accelerates from 4 to 40 rpm over the first 5 min of the trial and then remains at maximum speed for the remaining 5 min.Animals rested for at least 10 min between trails to avoid fatigue and exhaustion.Morris Water Maze Test: Over five days, animals were tested for four sessions daily.The hidden platform remained at a fixed spatial location for the entire acquisition period (5 days).Mice were released facing the wall of the maze at each session of the trial.Four equally distributed points along the wall served as starting points (N, S, E, and W) and divided the area into four quadrants.During the experiment, mice were constantly monitored and traced with a computer-assisted video tracking system (Noldus Ethovision).Elevated Plus Maze test: Mice were placed on the center platform of the maze, facing a closed arm, and allowed to explore the

Immunohistochemistry
Upon animal sacrifice, the cerebellum was collected and placed for 24 hours in a 10% formaldehyde solution, followed by incubation in 70% ethanol.After treatment with 0.3 % hydrogen peroxide, slides were briefly boiled in 10 mM sodium citrate, pH 6.0, for antigen enhancement.The sections were incubated with primary antibodies overnight at 4°C.Labeled sections were visualized with a Zeiss confocal microscope or the Keyence X800.
treatment induces the exchange of mitochondrial proteins between OMM and IMM.a, Western blot analysis of mitochondrial proteins' distribution between OMM (outer mitochondrial membrane), IMS (intermembrane space), and IMM (inner mitochondrial membrane) in UM-SCC-1A cells treated with vehicle (left) and SoSe (right).b-i, Quantification of a was done using Fiji software.Data are means SD (n=3, *p <0.05, **p<0.01,***p<0.001).The level of correspondent proteins in a whole lysate (WL) was used for normalization. in vitro activity assay.a, Mass-spectrometry analysis of 17C18- control and shICT1 cells.Actin was used as a loading control.j, Comparison of two CerS1 antibodies manufactured by Santa Cruz (# sc-65096) and MyBioSource (# MBS7104965) using WB analysis of total lysates obtained from Scr and CerS1 siRNA transfected 1A cells and treated with vehicle or SoSe. the mitochondrial membrane proteins.a, TEM images of mitochondria isolated from the UM-SCC-22A cells treated with 10 µM of SoSe and goldlabeled with anti-Tom20 antibody.Yellow arrows indicate the transition of Tom20 from the OMM at the beginning of the experiment (0 h) to IMM at the 3 h of SoSe exposure.Images represent at least three independent experiments.b, Identification of p17 ribosomal binding site.Multiple sequence alignment of ribosomal proteins from bacteria (L1 ribosomal protein [Methanococcus Jannaschii]; human (NP_000983.1 -60S ribosomal protein L29 [Homo sapiens]) and p17/PERMIT.The red box indicates sequences homologs to the p17 CerS1 binding site; the blue box marks sequences responsible for ribosome binding.To inhibit p17 interaction with ribosomes, the amino acids highlighted by the blue box (Phe 39, Leu 40, Arg 41, and Asn 42) have been replaced with Ala to generate a p17 mutant lacking ribosomal binding (p17Rb).c, CerS1 levels in mitochondria isolated from shp17 and shScr UM-SCC-1A cells transiently expressing EV, p17WT, and p17Rb mutant and treated with SoSe (10 µM, 3 h).COXIV, a mitochondrial marker, has been used as a loading control.Images represent at least three independent experiments.d, Quantification of c.Data are means ± SD (n=3 independent experiments, **p <0.01).e, left panel, Co-IP analysis of p17/CerS1 and p17/ICT1 interactions in UM-SCC-1A cells from c. e, right panel, Expression levels of p17 protein in Scr control cells and shp17 stable knocked out transiently transfected with empty vector (EV), p17WT, p17 mutant lacking ribosomal recognition sequence (p17Rbmut) and p17 mutant unable to interact with CerS1 (p17RYE/AAA).f, g, Quantification of e. Data are means ± SD (n=3 independent experiments, *p <0.05, **p <0.01) Topological analysis by Proteinase K digestion of TOM40 in 1A cells treated with SoSe for the indicated periods (top).Actin levels in the whole lysate were used as a normalization control.b, co-IP analysis of time-dependent interactions between Drp1-p17/PERMIT and p17/PERMIT-ICT1.c, Quantification of b. d, Live cell confocal microphotographs of 1A cells labeled with Mtphagy dye (red) and Lysotracker green (LTG, green) and treated for 3 hours with 5 mM of ULK1 inhibitor (SBI-0206965) or 1 mM Mdivi1 (Drp1 inhibitor) alone or in combination with 10 mM of SoSe.e, Quantification of d using Fiji software.Data are means SD (n=3, *p <0.05, **p<0.01,***p<0.001).f, ACO2, p-ULK1, and ULK-1 levels in cells from a. Actin was used as a loading control.g, Assessment of p17/PERMIT involvement in PARKIN independent mitophagic pathway.Whole lysate ACO2 levels in Scr control and sh-p17/PRRMIT SHSY-5Y cells and treated with PMI (10 mM for 24 h) and SoSe (10 mM, 3 h) (top).Bottom, mitochondrial levels of CerS1 in cells from the top panel.Actin and TOM20 were used as loading controls correspondently.h, Levels of p62 in SHSY-5Y cells treated with vehicle and p62-mediated mitophagy inductor (PMI, 10 mM 24 h).
of LCL768 on mitophagy induction in WT and p17/PERMIT-/-mice.a, Concentration of LCL768 at the indicated periods in serum, brain, kidney, and liver isolated from WT animals intravenously injected with LCL768.The concentration of LCL768 during indicated periods in serum (b) and kidney (c) of p17KO animals intracranially implanted with Alzet's pumps loaded with LCLC768.Data are means ± SD (n=3, **p <0.01).d, Lipids profile analysis of whole lysate and mitochondrial fractions isolated from cerebellums of 16 [15+28 days] months old p17KO animals implanted either with vehicle or LCL768 for 28 days.Data are means ± SD (n=3, **p <0.01).e, Levels of ACO2 and LC3 in whole lysates from b, c, f.Levels of ACO2 and LC3 proteins in the cerebellum lysates isolated from 15 months old WT and p17KO animals implanted with Alzets pumps loaded with vehicle (-) or LCL768 (+).Actin is used as a loading control.g, h, Quantification of f. i, Levels of LC3 and TOM40 interaction in cerebellum isolated from 15 months old p17KO animals implanted with Alzet' pumps loaded either with vehicle (-) or LCL7868 (+) measured by co-IP.j, Quantification of i.Data are means ± SD (n=3, *p <0.05).Interaction between LC3 and TOM40 (k) and LC3, TOM40, and PARKIN (m) was measured by co-IP in cerebellum isolated from animals from b. l, n Quantification of k and m correspondently.Data are means ± SD (*p>0.05).four hours.Alzet pumps' intracranial implantation and LCL768 treatment in mice: For the procedure, we used Alzet mini-osmotic pump model 2004 and brain infusion kit #3 according to the manufacturer's instructions (http//www.jove.com/vodeo/50326;DOI: doi:10.3791/50326).
apparatus for 5 minutes.Time spent in the open area, distance traveled in the open and closed parts, and the number of stretching postures were evaluated.Bright-Light Open Field Test: Mice were placed in the center of the open-field apparatus' brightly lit (200-300 lux) chamber (44x44x30 cm).An automatic monitoring system tracked the movements of the animals for 5 min.Horizontal motor (distance traveled) and main activity (distance traveled in central area/ total distance traveled) were evaluated.Novelty Preference test (Y-maze): This test included two trials.During Trial 1, one of the arms of the Y-maze was blocked, allowing for a three min exploration of only two arms of the maze.After a 1 min delay, Trial 2 was started.During Trial 2, all three arms were available for another 3 min of exploration.Trial 2 took advantage of the innate tendency of mice to explore novel unexplored areas (e.g., the previously blocked arm).The time spent in novel new areas of each animal was measured.Mice with intact short-term memory prefer to explore a novel arm over the familiar arms, whereas mice with impaired episodic memory enter all arms randomly.String Suspension test: Mice were permitted to grasp the string only by their forepaws and then released.Each mouse was tested three times (each test takes 20 sec) and scored from 0 to 3 depending on the severity of deficits.Ledge Assay: Mice were placed on the cage's ledge and monitored.Each mouse was tested two times (each test takes 20 seconds).If a mouse walked along the ledge and back into the cage delicately, the score of 0; if it fell off the ledge or avoided walking, a score of 3 was recorded.