Spatial selectivity of ATase inhibition in mouse models of Charcot–Marie–Tooth disease

Abstract The endoplasmic reticulum acetylation machinery has emerged as a new branch of the larger endoplasmic reticulum quality control system. It regulates the selection of correctly folded polypeptides as well as reticulophagy-mediated removal of toxic protein aggregates with the former being a particularly important aspect of the proteostatic functions of endoplasmic reticulum acetylation. Essential to this function is the Nε-lysine acetyltransferase activity of acetyltransferase 1 and acetyltransferase 2, which regulates the induction of endoplasmic reticulum–specific autophagy through the acetylation of the autophagy-related protein 9A. Here, we used three mouse models of Charcot–Marie–Tooth disease, peripheral myelin protein 22/Tr-J, C3-peripheral myelin protein 22 and myelin protein zero/ttrr, to study spatial and translational selectivity of endoplasmic reticulum acetyltransferase inhibitors. The results show that inhibition of the endoplasmic reticulum acetyltransferases selectively targets misfolding/pro-aggregating events occurring in the lumen of the organelle. Therefore, they establish acetyltransferase 1 and acetyltransferase 2 as the first proven targets for disease-causing proteotoxic states that initiate within the lumen of the endoplasmic reticulum/secretory pathway.


Introduction
A fundamental task of the endoplasmic reticulum (ER) is to make proteins that can then engage the secretory pathway to reach their final destination within the cell or be secreted to the extracellular milieu.Quality control mechanisms are in place to ensure that only correctly folded polypeptides can leave the ER.Quality control mechanisms are also in place to remove misfolded/unfolded polypeptides that would otherwise accumulate in the ER and cause proteotoxicity. 1,2y ensuring the continuous and efficient removal of toxic protein aggregates, the ER-specific autophagy (alternatively referred to as reticulophagy, ER-phagy or ER-associated degradation type II) represents a fundamental component of the ER quality control system.4][5][6] As such, it is not surprising that improving normal proteostatic mechanisms represents an active target for biomedical research.
The ER acetylation machinery has emerged as a novel branch of the larger ER quality control system.][18] The disposal of toxic protein aggregates through reticulophagy is an important aspect of the proteostatic functions of ER acetylation.This process requires ATase1-and ATase2-mediated acetylation of the ER-bound autophagy protein ATG9A, which in turn regulates the recruitment of the autophagy core machinery. 13,19In the mouse, reduced ER acetylation causes increased induction of reticulophagy, while increased ER acetylation has the opposite effect. 9,11,12enetic disruption or biochemical inhibition of the ATases results in activation of reticulophagy, as well as rescue of disease-associated proteotoxicity. 10,12,14,15,20Diseasecausing mutations in genes involved in the regulation of the proteostatic functions of the ER acetylation machinery (i.e.2][23][24][25][26] Finally, HSAN/ HMN-causing mutations have been associated with mislocalization of ATG9A and impaired autophagic degradation of pathogenic aggregates. 27,28n terms of translational output for autophagy-based strategies, a fundamental need is to selectively target autophagy to a specific cellular location.Studies conducted in different mouse models of proteotoxicity indicate that increasing reticulophagy through targeted inhibition of the ATases is a valid strategy to rescue disease-causing proteotoxic states of the ER and secretory pathway. 10,12,14,15,20owever, these studies were not designed to discriminate between disease-causing events that force a target protein to misfold/aggregate (i.e. a mutation) and disease-causing events that do not (i.e. a gene duplication) or between misfolding events that affect the luminal versus the cytosolic portion of ER-bound membrane proteins.This is a particularly important aspect of 'personalized medicine' since different genetic events (i.e.mutation, duplication or deletion) can be the underlying pathogenic mechanism.Sometimes, these different genetic events are associated with the same group of diseases (i.e.HSAN/HMN/SPGs) or even target the same protein (i.e.peripheral myelin protein 22, PMP22). 29,30ere, we used three mouse models of Charcot-Marie-Tooth disease, a peripheral form of neuropathy, to target the above limitations.The effectiveness of targeting ATase activity was tested using neuropathy models that involve duplication or misfolding mutations of two abundant peripheral myelin proteins in Schwann cells of peripheral nerves.The results show that ATase inhibition selectively targets misfolding/ pro-aggregating events occurring in the lumen of the ER, thus establishing the ATases as the first proven targets for proteotoxic states that initiate within the lumen of the ER.
Mice were housed and fed as described. 31,32The diet with Compound 9 (C9; 1 mg/g) was manufactured by Bio-Serv. 12,15,20Treatment with C9 began at weaning and continued throughout the entire life of the animals.All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison (protocol #M005120) and performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.Wild-type (WT) littermates were used as controls throughout the study.The age and sex of the animals at time of experimentation are specified in the figure and figure legends.Genotyping from tail DNA was performed at weaning by Transnetyx using real-time polymerase chain reaction.

Behaviour testing
All behavioural assays were conducted at the Waisman Center Behavioral Testing Service (Madison, WI, USA).All mice received a minimum of 30 min acclimation time to the testing room prior to each behaviour assay.

Phenotypic severity score
We used a simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia described previously. 33Blind analysis of the animals was performed for three different tests: ledge, hindlimb clasping and gait.Individual measures are scored on a scale of 0 to 3, with 0 representing an absence of the relevant phenotype and 3 representing the most severe manifestation (Supplementary Table 1).

Open field exploration
The specified protocol was performed according to Rigby et al. 31,32 Data were recorded using the Omnitech Fusion system.

Hot plate
Mice were placed on a hot plate (Columbus Instruments, hot plate analgesia metre) to evaluate the reaction time.The reaction time was scored when the animal jumped or licked its paws.A cut-off of 40 s was used to avoid any paw damage.Five reaction times were determined for each mouse with a latency of at least 15 min apart between measurements.The results shown for each animal are the average of the middle three values.

Grip strength
Grip strength was measured using an Ametek Chatillon DFE II (Columbus Instruments, grip strength metre).The mouse was held by the base of the tail above a wire bar connected to the force gauge.The mouse was placed in a position that allowed it to grasp the wire with its forepaws and then pulled away from the bar at a constant speed.The maximum force generated just before the mouse lost its grasp was recorded.This was repeated five times for each animal.The results shown for each animal are the average of the middle three values.

Inverted screen
Mice were removed from the home cage and placed on top of the screen.A gentle shake of the screen was performed to make sure the mouse had gripped the screen.Then, the screen was carefully inverted at 30 cm over the empty cage so that the mouse was upside down on the screen.Time was measured from the inversion moment to record the time latency to fall.This was repeated five times for each animal with a latency of at least 10 min between measurements.The results shown for each animal are the average of the middle three values.

Balance beam
The balance beam apparatus is composed of one smooth, plastic beam 70 cm in length and 3 cm in diameter.The beam is securely suspended 25 cm above the surface.Enclosed safe house is placed at the escape end of the beam, and bedding is added to encourage the mouse to enter.A training session was conducted the day before the test session.The test session consists of five runs per animal, and the results are shown as total successful runs per animal (0-5 score).

Electron microscopy
Following CO 2 euthanasia, sciatic nerves were extracted and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C.The specified protocol involving fixation, dehydration, embedding and sectioning was performed according to Peng et al. and Pehar et al. 9,34 The sectioned samples were viewed at 80 kV on a Philips CM120 transmission electron microscope equipped with AMT BioSprint12 digital camera (AMT Imaging Systems).

Morphometry
Non-overlapping electron micrographs of sciatic nerves were analysed for axon diameter and g-ratio.A minimum of 100 randomly selected fibers were analysed per animal using the g-ratio plug-in of the ImageJ software, which allowed for semi-automated analysis of randomly selected sets of fibres. 35For each nerve, the percentage of non-myelinated axons was calculated by direct count from the electron microscopy micrographs.

Statistics and reproducibility
No statistical method was used to determine the necessary sample size for each experiment.The number of experimental replicates, representing the number of mice per genotype, is indicated in the respective legends.Data analysis was performed using GraphPad Prism version 9.5.1.733.Data are expressed as mean ± standard deviation.Comparison of the means was performed using an unpaired t-test for two groups and ordinary one-way or two-way ANOVA for ≥3 groups followed by Tukey-Kramer (comparison between all groups) multiple comparison test.Statistical details are described in the figure legends.Differences were declared statistically significant if P < 0.05.The following statistical significance indicators are used: *P < 0.05, **P < 0.005 and # P < 0.0005.

Results
To determine the ability of ATase inhibitors to discriminate between disease states characterized by ER proteotoxicity and those that are not, we targeted three mouse models of Charcot-Marie-Tooth disease, Pmp22 Tr−J , C3-PMP22 and Mpz ttrr .Although they all develop a peripheral form of neuropathy that resembles Charcot-Marie-Tooth disease, the genetic and molecular underpinnings are very different.7][38][39][40][41][42] Consistently, when expressed in Chinese Hamster Ovary (CHO) cells, the Tr-j/trembler Jackson mutant version of human PMP22 (PMP22 Tr-J ) displayed a punctate staining that appeared to be fully sequestered in the ER while the WT version (PMP22 WT ) did not (Supplementary Fig. 1).In contrast to PMP22-based mice, Mpz ttrr mice carry a spontaneous mutation of mouse Mpz.Both PMP22 and MPZ are membrane proteins that insert into the ER membrane.However, the Tr-j mutation on PMP22 resides in the lumen of the ER while the ttrr/totterer mutation on MPZ resides on the cytosolic portion of the protein (Supplementary Fig. 2).When expressed in CHO cells, the ttrr mutant version of human MPZ (MPZ ttrr ) appeared mislocalized, as compared with the WT version (MPZ WT ), but did not display any sequestration within the ER (Supplementary Fig. 1).This finding is consistent with the mislocalization of other C-terminal alterations of MPZ. 43inally, to separate soluble and aggregated species of both mutants, MPZ ttrr -and PMP22 Tr-J -expressing cells were sequentially lysed with Triton ™ X-100 (soluble protein species) and sodium dodecyl sulfate (aggregated protein species), and the mutant proteins were analysed based on their migration profile.In contrast to MPZ ttrr , PMP22 Tr-J was almost exclusively found in the aggregated form (Supplementary Fig. 3).Therefore, Pmp22 Tr−J , C3-PMP22 and Mpz ttrr mice can be used as proof of concept to establish the selectivity of ATase1/ATase2 inhibitors towards ER-specific proteotoxicity.
Pmp22 Tr−J mice were treated with an oral formulation (50 mg/kg/day) of C9, a specific inhibitor of ATase1 and ATase2, following the well-established administration protocols. 10,12,15,20Treatment began at weaning (post-natal day 21), a time when the mice already displayed phenotypic deficits.The evolution of the disease phenotype was initially determined with a modified ataxia severity score (Supplementary Table 1), which examines ledge wall, hindlimb clasping and gait functions. 33,44,45As expected, Pmp22 Tr−J mice scored very poorly with a combined severity score in the 6-8 range in both sexes while WT remained stable within the 0-2 range (Fig. 1A).C9 treatment reduced the severity score in both sexes (Fig. 1A).Importantly, the improvement remained stable throughout the entire period of treatment and was manifested across all measured functions (Fig. 1A-1C; Supplementary Fig. 4).Next, we evaluated the animals for total distance travel and rear/hind paws standing frequency on the open field test battery.In both cases, C9 treatment produced a consistent and stable improvement, which was evident in both males and females and throughout the entire period of treatment (Fig. 2A and B).
Post-mortem electron micrograph evaluation of the sciatic nerve revealed a marked loss of myelin in Pmp22 Tr−J mice (Fig. 3A).This was partially prevented by C9 treatment (Fig. 3A).Importantly, C9 treatment reduced the number of unmyelinated axons by about 40-50% (Fig. 3B) and increased the thickness of myelin sheets around myelinated axons, as manifested by the ratio of inner-to-outer axonal diameter (g-ratio; Fig. 3C and D).Again, the g-ratio rescuing effect remained stable throughout the entire period of treatment.A salient feature of C9 treatment was the restoration of myelin around smaller diameter axons (Fig. 3A; Supplementary Fig. 5).
In contrast to Pmp22 Tr−J mice, ATase inhibition of C3-PMP22 or Mpz ttrr mice did not elicit phenotypic improvement (Figs. 4 and 5).The Charcot-Marie-Tooth disease-like phenotype of Mpz ttrr mice developed earlier than C3-PMP22 mice and was much more severe, with marked defects across different motor-based paradigms.The different phenotypic severity was manifested at the histological level with Mpz ttrr mice displaying a drastic loss of neurons and myelin (Figs. 4 and 5).Importantly, no rescuing effect was observed following treatment with C9 (Figs. 4 and 5).
In conclusion, the inhibition of the ATases by C9 partially rescued both the behavioural and pathological features associated with the Charcot-Marie-Tooth diseaselike phenotype of Pmp22 Tr−J mice but was not able to modify the progression of the disease in C3-PMP22 or Mpz ttrr mice.

Discussion
ATase1 and ATase2 are type II ER-resident membrane proteins with the catalytic domain facing the lumen of the organelle. 18They use acetyl-CoA, imported into the ER lumen by AT-1/SLC33A1, to acetylate ER cargo and resident proteins. 17Genetic disruption of either Atase1 or Atase2 in the mouse stimulates reticulophagy. 14Biochemical inhibition of the Atases in the mouse also stimulates reticulophagy. 10,15,20Finally, reduced import of acetyl-CoA into the ER lumen stimulates reticulophagy by limiting the catalytic activity of the ATases. 9,10,17,46Both cell-and animal-based studies indicate that the regulation of reticulophagy downstream of the ATases depends on the acetylation status of ATG9A.14]19 In essence, the ER acetylation machinery appears to be spatially positioned to act as a novel branch of the ER quality control system to help disposing of protein aggregates that form within the ER lumen.
Inhibition of the ATases was able to rescue the progerialike phenotype of AT-1 sTg and SLC13A5 sTg, two mouse models of ER hyperacetylation. 12,15,20Inhibition of the ATases was also able to resolve the Alzheimer's diseaselike phenotype of APP 695/swe and APP 695/swe /PS1-dE9 mice. 10,20Importantly, ATase inhibition did not resolve the disease phenotype of mHtt Q160 mice, a model of Huntington disease, or hSOD1 G93A mice, a model of amyotrophic lateral sclerosis. 10APP is a type I membrane protein that inserts into the ER to engage the secretory pathway, while Htt and SOD1 are cytosolic proteins and do not engage the secretory pathway.Furthermore, the proteotoxic aggregates of the A53T mutant version of α-synuclein, which is associated with an autosomal dominant form of Parkinson's disease, were successfully degraded by reduced ER acetylation only when α-synuclein was forced to insert into the ER lumen by adding a signal peptide to its N-terminus. 10In essence, ATase inhibitors appear to be selective for ER/secretory pathway proteotoxicity.This study adds an additional layer of evidence by showing that misfolding states that occur in the lumen of the ER, and that are associated with aberrant aggregation of the misfolded polypeptide in the ER, can be targeted by ATase inhibitors, while misfolding states that occur elsewhere, or that do not yield protein aggregates within the ER, cannot.In essence, work conducted with different mouse models of human diseases has established the ATases as the first proven targets for proteotoxic states that initiate within the lumen of the ER (see present study and Peng et al., Rigby et al., Fernandez-Fuente et al. and Murie et al. 10,12,14,15,20 ).This selectivity provides encouraging hopes for many hereditary diseases caused by mutations that force the protein to misfold and aggregate within the ER and secretory pathway, such as Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease and cystic fibrosis.It also provides a way to limit or completely avoid unwanted effects caused by non-selective global activation of autophagy.This selectivity, however, also requires a personalized form of medicine with careful selection of patients, particularly for those diseases, such as hereditary forms of neuropathy, where different genetic events (i.e.mutation, duplication or deletion) can be the underlying pathogenic mechanism.Using Charcot-Marie-Tooth disease as a test case, our data would indicate that ATase inhibition may not be effective in patients with PMP22 duplication (classified as CMT1A) but may be effective in patients with PMP22 mutations (classified as CMT1E).Although treatment of mice with the Mpz ttrr mutation was ineffective, there are a variety of dominant MPZ mutations (classified as CMT1B) that are associated with the misfolding of the ER luminal portion of the protein, ER retention and even ER aggregation together with activation of the unfolded protein response. 47,48These forms of Charcot-Marie-Tooth disease

Figure 1
with single point graphs is shown as Supplementary Fig.4.

Figure 3
Figure 3 ATase inhibition improves myelin morphology in the Pmp22 Tr−J mice.(A) Electron micrographs of the sciatic nerves at 5 months of Pmp22 Tr−J , Pmp22 Tr−J treated and WT

Figure 4
Figure 4 Phenotypic assessment of C3-PMP22 mice.(A) Phenotypic severity score represented as a sum of ledge, gait and hindlimb clasping at different ages (n = 7/group).**P < 0.005 and