Depletion of Paraoxonase 1 (Pon1) Dysregulates mTOR, Autophagy, and Accelerates Amyloid Beta Accumulation in Mice

Paraoxonase 1 (PON1), a homocysteine (Hcy)-thiolactone detoxifying enzyme, has been associated with Alzheimer’s disease (AD), suggesting that PON1 plays an important protective role in the brain. To study the involvement of PON1 in the development of AD and to elucidate the mechanism involved, we generated a new mouse model of AD, the Pon1−/−xFAD mouse, and examined how Pon1 depletion affects mTOR signaling, autophagy, and amyloid beta (Aβ) accumulation. To elucidate the mechanism involved, we examined these processes in N2a-APPswe cells. We found that Pon1 depletion significantly downregulated Phf8 and upregulated H4K20me1; mTOR, phospho-mTOR, and App were upregulated while autophagy markers Bcln1, Atg5, and Atg7 were downregulated at the protein and mRNA levels in the brains of Pon1─/─5xFAD vs. Pon1+/+5xFAD mice. Pon1 depletion in N2a-APPswe cells by RNA interference led to downregulation of Phf8 and upregulation of mTOR due to increased H4K20me1-mTOR promoter binding. This led to autophagy downregulation and significantly increased APP and Aβ levels. Phf8 depletion by RNA interference or treatments with Hcy-thiolactone or N-Hcy-protein metabolites similarly increased Aβ levels in N2a-APPswe cells. Taken together, our findings define a neuroprotective mechanism by which Pon1 prevents Aβ generation.


Introduction
Paraoxonase 1 (PON1), named for its ability to hydrolyze and inactivate the organophosphate paraoxon, is synthesized exclusively in the liver, circulates in the blood as a component of high-density lipoproteins (HDL) [1], and is present in many organs, including the brain [2]. In addition to protecting from organophosphate toxicity [3], PON1 protects against atherosclerosis induced by a high-fat diet [4] or ApoE depletion [5] in mice. Large-scale human studies showed that high arylesterase activity of PON1 protects from cardiovascular disease (CVD) in patients with coronary artery disease undergoing elective diagnostic coronary angiography [6,7] and in patients with chronic kidney disease [8], while low homocysteine thiolactonase activity of PON1 was associated with worse longterm mortality [9]. In the PREVEND prospective study involving 6902 participants, PON1 activity predicted CVD events [10]. The cardioprotective function of PON1 can be due both to its antioxidative function [4,6,11] and the ability to detoxify homocysteine (Hcy)thiolactone [12][13][14][15], thereby attenuating lipid peroxidation, oxidative protein modification, and protein N-homocysteinylation.
PON1 has also been implicated in Alzheimer's disease (AD) [16,17], which can be expected given that AD has a significant vascular component [18]. For example, PON1 activity is lower in AD and dementia patients compared with healthy controls [19][20][21][22] and correlates with the severity of AD-related cognitive decline [23]. In patients with mild cognitive impairment, PON1 activity predicted global cognition, verbal episodic memory, and attention/processing speed [24]. In mice, ApoE −/− Pon1 −/− animals, which have severe carotid atherosclerosis [5], showed AD markers and impaired vasculature in their brains at 14 months, although it was not clear whether brain pathology was caused by ApoE −/− , Pon1 -/-, or both knockouts [25]. In a mouse model of AD (Tg2576), immunohistochemical fluorescence signals for Pon1 protein in various regions of the brain were found to surround Aβ plaques but could not be colocalized to any brain cell type [26].
Deletion of the Pon1 gene in mice impairs the metabolic conversion of Hcy-thiolactone to Hcy, increases brain Hcy-thiolactone levels, and makes the animals overly sensitive to the neurotoxicity of Hcy-thiolactone injections [12]. Studies of Pon1 −/− mouse brain proteome demonstrated that Pon1 interacts with diverse cellular processes, such as energy metabolism, anti-oxidative defenses, cell cycle, cytoskeleton dynamics, and synaptic plasticity, that are essential for brain homeostasis [27]. Clusterin (CLU or APOJ), involved in the transport of amyloid beta (Aβ) from plasma to brain in humans (reviewed in [28]), is carried on a distinct HDL subspecies that contains three major proteins: PON1, CLU, and APOA1 [29]. Notably, levels of Clu (ApoJ) are significantly elevated in the plasma of Pon1 −/− vs. Pon1 +/+ mice [30]. These findings suggest that Pon1 plays a key role in brain homeostasis, possibly protecting from Aβ accumulation.
The present work was undertaken to examine the effects of Pon1 depletion on Aβ levels in a novel model of AD, the Pon1 -/-5xFAD mouse, generated in the present study and to elucidate the mechanism involved. Because dysregulated mTOR signaling and autophagy have been implicated in Aβ accumulation in Alzheimer's disease [31,32], and H4K20me1 demethylation by PHF8 is important for maintaining homeostasis of mTOR signaling [33], we studied how these processes are affected by Pon1 depletion in the mouse neuroblastoma N2a-APPswe cells and Pon1 -/-5xFAD mice. We also examined how changes in these processes affect the behavioral performance of Pon1 -/-5xFAD mice.
Water supplemented with 1% methionine was used to induce hyperhomocysteinemia [12,27]. The high Met diet increases plasma total Hcy levels 5.6-and 10.4-fold in Pon1 −/− (from 8.5 to 48 µM) and Pon1 +/+ mice (from 7.4 to 77 µM) [27]. Animal procedures were approved by the Institutional Animal Care and Use Committee at the New Jersey Medical School.

Brain Protein Extraction
Mice were euthanized by CO 2 inhalation; the brains were collected and frozen on dry ice. Frozen brains were pulverized with dry ice using a mortar and pestle and stored at −80 • C. Proteins were extracted from the pulverized brains (50 ± 5 mg; 30 ± 3 mg brain was used for Aβ analyses) using RIPA buffer (4 v/w, containing protease and phosphatase inhibitors) with sonication (Bandelin SONOPLUS HD 2070) on wet ice (three sets of five 1-s strokes with 1 min cooling interval between strokes). Brain extracts were clarified by centrifugation (15,000× g, 30 min, 4 • C) and clear supernatants containing 8-12 mg protein/mL were collected (RIPA-soluble fraction). Protein concentrations were measured with BCA kit (Thermo Fisher Scientific, Waltham, MA, USA).

Aβ Quantification
Aβ was quantified using a dot blot assay [36]. Briefly, brain protein extracts (1 µL) were spotted onto the nitrocellulose membranes and dried (37 • C, 1 h). The membranes were washed with TBST buffer (RT, 15 min) and blocked with 5% BSA in TBST buffer (RT, 1 h). Blocked membranes were washed three times with TBST buffer (10 min each) and incubated with monoclonal anti-Aβ antibody (CS #8243; 4 • C, 16 h). Membranes were then washed three times with TBST buffer (10 min each) and incubated with goat horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. Positive signals were detected using Western Bright Quantum-Advansta K12042-D20 and GeneGnome XRQ NPC chemiluminescence detection system. Signal intensity was assessed using the Gene Tools program from Syngene.
For gene silencing, siRNAs targeting the Pon1 (Cat. # s71950 and s71951) or Phf8 gene (Cat. # s115808, and s115809) (Thermo Scientific) were transfected into cells maintained in Opti-MEM medium by 48-h Lipofectamine RNAiMax (Thermo Scientific) treatments. Cellular RNA for RT-qPCR analysis was isolated as described in Section 2.5. For protein extraction, RIPA buffer (MilliporeSigma) was used according to the manufacturer's protocol.

Western Blots
Proteins were separated by SDS-PAGE on 10% gels (20 µg protein/lane) and transferred to a PVDF membrane (Bio-Rad) for 20 min at 0.1 A, 25 V using the Trans Blot Turbo Transfer System (Bio-Rad). After blocking with 5% bovine serum albumin in TBST buffer (1 h, room temperature), the membranes were incubated with monoclonal anti-Pon1 Membranes were washed three times with TBST buffer, for 10 min each, and incubated with goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase. Positive signals were detected using Western Bright Quantum-Advansta K12042-D20 and GeneGnome XRQ NPC chemiluminescence detection system. Band intensity was calculated using the Gene Tools program from Syngene.
2.6. RNA Isolation, cDNA Synthesis, RT-qPCR Analysis Total RNA was isolated using Trizol reagent (MilliporeSigma). cDNA synthesis was conducted using Revert Aid First cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Nucleic acid concentration was measured using NanoDrop (Thermo Fisher Scientific). RT-qPCR was performed with SYBR Green Mix and CFX96 thermocycler (Bio-Rad, Hercules, CA, USA). The 2 (−∆∆Ct) method was used to calculate the relative expression levels [39]. Data analysis was performed with the CFX Manager™ Software, Microsoft Excel, and Statistica. RT-qPCR primer sequences are listed in Table S1.

Chromatin Immunoprecipitation Assay
For CHIP assays we used CUT&RUN Assay Kit #86652 (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer's protocol. Each ChIP assay was repeated three times. Briefly, for each reaction, we used 100,000 cells. Cells were trypsinized and harvested, washed 3× in ice-cold PBS, and bound to concanavalin A-coated magnetic beads for 5 min, at RT. Cells were then incubated (4 h, 4 • C) with 2.5 µg of anti-PHF8 antibody (Abcam, ab36068) or anti-H4K20me1 antibody (Abcam, ab177188) in the antibody-binding buffer plus digitonin that permeabilizes cells. Next, cells were treated with pAG-MNase (1 h, 4 • C), washed, and treated with CaCl 2 to activate DNA digestion (0.5 h, 4 • C). Cells were then treated with the stop buffer and spike-in DNA was added for each reaction for signal normalization, and incubated (10-30 min, 37 • C). Released DNA fragments were purified using DNA Purification Buffers and Spin Columns (CS #14209) and quantified by RT-qPCR using primers targeting the promoter, upstream, and downstream regions of the mTOR gene (Table S1). Rabbit (DA1E) mAb IgG XP ® Isotype Control included in the CUT&RUN kit did not afford any signals in the RT-qPCR assays targeting mTOR.

Confocal Microscopy, Aβ Quantification in N2a-APPswe Cells
Mouse neuroblastoma N2a-APPswe cells were cultured in Millicell EZ SLIDE 8-well glass slides (Merck, Darmstadt, Germany). After treatments, cells were washed 3 times with PBS for 10 min. Cells were fixed with 4% PFA (MilliporeSigma) (37 • C, 15 min), washed 3 times with PBS buffer, permeabilized with 0.1% Triton X-100 solution (RT, 20 min), blocked with 0.1% BSA (RT, 1h), and incubated with anti-Aβ antibody (CS #8243; 4 • C, 16 h). Cells were then washed 3 times with PBS and stained with secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) (Abcam, ab150077; RT, 1 h) to visualize and quantify Aβ. DAPI (Vector Laboratories, Newark, CA, USA) was used to visualize nuclei. Fluorescence signals were monitored by using a Zeiss LSM 880 confocal microscope with a 488 nm filter for the Alexa Fluor ® 488 (Aβ) and 420-480 nm filter for DAPI, taking a z stack of 20-30 sections with an interval of 0.66 µm and a range of 15 µm. Zeiss Plan-Apochromat X40/1.2 Oil differential interference contrast objective were used for imaging. Images were quantified with the ImageJ Fiji 2.9.0 software (NIH, Bethesda, MD, USA). The hindlimb clasping test is used to assess neurodegeneration in mouse models [40]. For this test, mice were suspended by the base of the tail and videotaped for 10 s. Three separate trials were taken over three consecutive days. Hindlimb clasping was scored from 0 to 3: 0 = hindlimbs splayed outward and away from the abdomen; 1 = one hindlimb retracted inwards towards the abdomen for at least 50% of the observation period; 2 = both hindlimbs partially retracted inwards towards the abdomen for at least 50% of the observation period; and 3 = both hindlimbs completely retracted inwards towards the abdomen for at least 50% of the observation period. Hindlimb clasping scores were added together for the three separate trials.

Ledge Test
The ledge test is used to assess motor deficits in rodent models of CNS disorders [41]. Typically, mice walk along the ledge of a cage and try to descend back into the cage. Three separate trials were taken for each mouse. The ledge test was scored from 0 to 3 points: 0 = a mouse walked along the ledge without slipping and lowered itself back into the cage using paws; 1 = the mouse lost its footing during walking along the ledge but otherwise appeared coordinated; 2 = the mouse did not effectively use its hind legs and landed on its head rather than paws when descending into the cage; and 3 = the mouse fell of the ledge or was shaking and/or barely moving.

Cylinder Test
The cylinder test is used to assess sensorimotor function in rodent models of CNS disorders. A mouse is placed in a transparent 500 mL plastic cylinder. The number of times the mouse rears up and touches the cylinder wall during a period of 3 min is counted. A rear is defined as a vertical movement with both forelimbs off the floor so that the mouse is standing only on its hindlimbs. At the end of 3 min, the mouse was removed and placed back into its home cage. Because spontaneous activity in the cylinder is affected by repeated testing, resulting in reduced activity over time, mice were tested only once in their lifetime.

Statistical Analysis
The results were calculated as mean ± standard deviation. A two-sided unpaired t test was used for comparisons between two groups of variables; p < 0.05 was considered significant. Statistical analysis was performed using Statistica, Version 13 (TIBCO Software Inc., Palo Alto, CA, USA, http://statistica.io) (accessed 2 November 2022).

Pon1 Depletion Upregulates mTOR and Inhibits Autophagy in Mouse Brain
Because Phf8/H4K20me1 regulate mTOR signaling, we next examined the effects of Pon1 depletion on levels of mTOR and its active form, phosphorylated at Ser2448 (pmTOR). We found that mTOR protein was significantly upregulated in the brains of
Overall, the effects of the Pon1 −/− genotype on mTOR and pmTOR levels were attenuated by the HHcy diet ( Figure 1C,D). These findings indicate that Pon1 depletion upregulated pmTOR to a similar extent as mTOR, suggesting that the Pon1 -/genotype affects mTOR signaling mostly at the level of mTOR protein expression.
These findings indicate that the Pon1 gene exerts transcriptional control over the expression of Phf8, mTOR, autophagy-related proteins, and APP in the mouse brain.

Pon1 Gene Silencing Downregulates the Histone Demethylase Phf8, Upregulates H4K20me1 Epigenetic Mark, mTOR and pmTOR, APP, and Inhibits Autophagy in Mouse Neuroblastoma N2a-APPswe Cells
To elucidate the mechanism by which Pon1 depletion impacts Phf8 and its downstream effects on mTOR, autophagy, and APP, we first examined whether the findings in Pon1 −/− mice can be recapitulated in cultured mouse neuroblastoma N2a-APPswe cells that overproduce Aβ from a mutated human APP transgene [38]. We silenced the Pon1 gene in these cells by RNA interference using Pon1-targeting siRNA and studied how the silencing impacts Phf8 and its downstream effects. Changes in specific protein levels in Pon1-silenced and control cells were analyzed by Western blotting using Gapdh protein as a reference.
The Western blot results show that the changes in Phf8, H4K20m31, mTOR signaling, autophagy, and APP induced by Pon1 gene silencing in N2a-APPswe cells (Figure 3) recapitulate the in vivo findings in the Pon1 −/− 5xFAD mouse brain (Figures 1 and 2).
The Western blot results show that the changes in Phf8, H4K20m31, mTOR signaling, autophagy, and APP induced by Pon1 gene silencing in N2a-APPswe cells (Figure 3) recapitulate the in vivo findings in the Pon1 −/− 5xFAD mouse brain (Figures 1 and 2).

Pon1 Gene Silencing Increases H4K20me1 Biding to mTOR Promoter in N2a-APPswe Cells
To determine whether increased levels of the histone H4K20me1 mark can promote mTOR gene expression by binding to its promoter in Pon1-depleted cells, we carried out ChIP experiments using anti-H4K20me1 antibody (Figure 4). The Pon1 gene was silenced by transfecting N2a-APPswe cells using two different Pon1-targeting siRNAs. The cells were permeabilized and treated with anti-H4K20me1 antibody and a recombinant micrococcal nuclease-protein A/G. DNA fragments released form N2a-APPswe cells were quantified by RT-qPCR using primers targeting the transcription start site (TSS) of the mTOR gene as well as upstream (UP) and downstream (DOWN) regions. We found that in Pon1-silenced N2a-APPswe cells, the binding of H4K20me1 was significantly increased at the mTOR TSS (1.8 to 2.3-fold, p = 4 × 10 −5 ), mTOR UP (2.0 to 2.2fold, p = 2 × 10 −5 ), and mTOR DOWN sites (1.4 to 1.6-fold, p = 1 × 10 −4 ) ( Figure 4A). Importantly, in Pon1-silenced cells there were significantly more DNA fragments from the mTOR TSS (2.3 ± 0.2 and 1.8 ± 0.2 for siRNA Pon1 #1 and #2, respectively) than from the DOWN site (1.4 ± 0.2 and 1.6 ± 0.1 for siRNA Pon1 #1 and #2, respectively; p = 0.004). There were also more DNA fragments from the UP site than from the DOWN site (2.2 ± 0.2 and 2.1 ± 0.2 for siRNA Pon1 #1 and #2 vs. 1.4 ± 0.2 and 1.6 ± 0.1 for siRNA Pon1 #1 and #2; p = 0.0004). Numbers of DNA fragments from the TSS and UP sites were similar (p = 0.713) ( Figure 4A). Control experiments showed that the binding of H3K4me3 to RPL30 intron was not affected by Pon1 gene silencing ( Figure 4B). These findings indicate that Pon1 gene We found that in Pon1-silenced N2a-APPswe cells, the binding of H4K20me1 was significantly increased at the mTOR TSS (1.8 to 2.3-fold, p = 4 × 10 −5 ), mTOR UP (2.0 to 2.2-fold, p = 2 × 10 −5 ), and mTOR DOWN sites (1.4 to 1.6-fold, p = 1 × 10 −4 ) ( Figure 4A). Importantly, in Pon1-silenced cells there were significantly more DNA fragments from the mTOR TSS (2.3 ± 0.2 and 1.8 ± 0.2 for siRNA Pon1 #1 and #2, respectively) than from the DOWN site (1.4 ± 0.2 and 1.6 ± 0.1 for siRNA Pon1 #1 and #2, respectively; p = 0.004). There were also more DNA fragments from the UP site than from the DOWN site (2.2 ± 0.2 and 2.1 ± 0.2 for siRNA Pon1 #1 and #2 vs. 1.4 ± 0.2 and 1.6 ± 0.1 for siRNA Pon1 #1 and #2; p = 0.0004). Numbers of DNA fragments from the TSS and UP sites were similar (p = 0.713) ( Figure 4A). Control experiments showed that the binding of H3K4me3 to RPL30 intron was not affected by Pon1 gene silencing ( Figure 4B). These findings indicate that Pon1 gene silencing induces H4K20me1 binding at the mTOR gene, significantly higher at the mTOR TSS and UP site than at the DOWN site in Pon1-silenced cells.
CHIP experiments using anti-Phf8 antibody showed that Pon1 gene silencing or treatments with Hcy-thiolactone or N-Hcy-protein did not affect binding of Phf8 to the mTOR gene.
Because Pon1 depletion elevates Hcy-thiolactone and N-Hcy-protein in mice [12], we examined whether any of these metabolites can induce Aβ accumulation in N2a-APPswe cells. In cells treated with Hcy-thiolactone (20-200 µM) or N-Hcy-protein (10-20 µM), there was significantly more Aβ, manifested by significantly increased area of fluorescent Aβ puncta in confocal immunofluorescence images compared with control-siRNA and siRNAscr ( Figure 5C,D). However, while treatments with Hcy-thiolactone led to increased size and signal intensity of the fluorescent Aβ puncta, treatments with N-Hcy-protein did not ( Figure 5D), suggesting different effects of Hcy-thiolactone and N-Hcy-protein on the structure of Aβ deposits. These findings suggest that Hcy-thiolactone and N-Hcy-protein contribute to elevated Aβ levels induced by Pon1 gene silencing.

Pon1 Depletion Increases Aβ Accumulation in Pon1 −/− 5xFAD Mice
Aβ was extracted from brains of 5-and 12-month-old mice fed with a standard chow diet, and from 5-month-old mice with the HHcy diet (1% Met in drinking water) since weaning at the age of 1 month. SDS-soluble and formic acid (FA)-soluble Aβ fractions, which contain the bulk of Aβ [36], as well as a minor RIPA-soluble Aβ fraction were obtained. Aβ was quantified in these fractions by a dot blot assay with a monoclonal anti-Aβ antibody [37].

Pon1 Depletion Does Not Induce Sensorimotor Deficits
To examine the effects of Pon1 depletion on neurodegeneration and sensorimotor activity, 12-month-old Pon1 −/− 5xFAD mice and their Pon1 +/+ 5xFAD sibling controls were assessed in the hindlimb clasping, ledge, and cylinder tests.

Discussion
In previous studies, we found that Pon1 is a Hcy-thiolactone-hydrolyzing enzyme [13] and that Pon1 depletion in mice elevated brain Hcy-thiolactone and N-Hcy-protein [12], increased the animals' susceptibility to Hcy-thiolactone-induced seizures [12], and resulted in pro-neurodegenerative changes in brain proteome [27], suggesting that Pon1 plays an important protective role in brain homeostasis.
Our present findings show that Pon1 protects from amyloidogenic APP processing to Aβ in mice brains ( Figure 6) and unravel the mechanistic basis of the protective role of Pon1 in the CNS. Specifically, we found that Pon1 depletion downregulated histone demethylase Phf8 both at the protein and mRNA level, increased H4K20me1 binding at the mTOR promotor ( Figure 4A), and upregulated mTOR expression and phosphorylation in the mouse brain ( Figure 1C,D) and neuroblastoma N2a-APPswe cells ( Figure 3D,E). Treatments with Hcy-thiolactone and N-Hcy-protein, metabolites that are elevated in Pon1 −/− mice, also increased H4K20me1 binding at the mTOR promotor in N2a-APPswe cells ( Figure 4C). This suggests that Pon1 is a negative regulator of mTOR signaling by controlling levels of Hcy metabolites that affect binding of H4K20me1 at the mTOR promotor. The effects of Hcythiolactone and N-Hcy-protein on mTOR are explained by findings that Phf8, the regulator of mTOR expression, was downregulated by Pon1 depletion (Figures 1A and 2A), whereas H4K20me1 was upregulated ( Figures 1B and 2A). These findings provide direct mechanistic evidence linking Hcy-thiolactone and N-Hcy-protein with dysregulated mTOR signaling and its downstream consequences such as downregulation of autophagy and upregulation of Aβ. This mechanism is further supported by our findings that Phf8 depletion by RNA interference affected mTOR, autophagy, APP, and Aβ, similar to treatments with Hcythiolactone or N-Hcy-protein [42] In the present study, we found that depletion of Pon1 upregulated APP in the Pon1 −/− 5xFAD mouse brain ( Figures 1H, 2 and S1F) and in mouse neuroblastoma N2a-APPswe cells ( Figure 3I). In contrast, depletion of Phf8 did not affect APP expression [42]. These findings suggest that Pon1 interacts with APP in the Pon1 −/− 5xFAD mouse brain while Phf8 does not. However, whether the Pon1-APP interaction is direct or indirect remains to be determined.
Although Pon1 depletion in mouse neuroblastoma N2a-APPswe cells downregulated Phf8 (Figures 1A and 2A) and upregulated APP (Figures 1H and 2A) and Aβ ( Figure 5), depletion of Phf8 upregulated Aβ but not APP [42]. These findings suggest that two pathways can lead to increased Aβ generation in Pon1-depleted brains and neural cells. One pathway involves Hcy metabolites, which upregulate APP, while another pathway involves impaired Aβ clearance due to downregulated autophagy.
Notably, Pon1 depletion caused changes in the Phf8-> H4K20me1-> mTOR-> autophagy pathway akin to the changes induced by HHcy in the mouse brain ( Figure 1) and neuroblastoma cells (Figure 3). Pon1 depletion or HHcy similarly increased accumulation of Aβ in the brain ( Figure 6). Our previous work showed that a common primary biochemical outcome of Pon1 depletion or of HHcy was essentially the same: HHcy caused elevation of Hcy-thiolactone and N-Hcy-protein [43] as did Pon1 depletion [12,14]. In the present work, Pon1 depletion by RNA interference or treatments with Hcy-thiolactone or N-Hcy-protein similarly increased the accumulation of Aβ in mouse neuroblastoma cells ( Figure 5). Taken together, these findings suggest that increased accumulation of Aβ in Pon1-depleted brains is mediated by the effects of Hcy metabolites on mTOR signaling and autophagy.
5xFAD mice develop sensorimotor deficits beginning at about 9 months of age (https: //www.alzforum.org/research-models/5xfad-b6sjl) (accessed 27 December 2022). For example, 5xFAD mice perform worse than the wild-type animals in the hindlimb and balance beam tests [44,45]. We found that depletion of Pon1 did not aggravate these deficits: there was no difference in sensorimotor performance between Pon1 −/− 5xFAD mice vs. Pon1 +/+ 5xFAD animals in the hindlimb, ledge, and cylinder tests ( Figure S2). These findings suggest that upregulated Aβ accumulation may not be causing sensorimotor impairment. However, other aspects of sensorimotor abilities may be affected by Pon1, which remains to be assessed in future studies, as are the effects of Pon1 on various domains of cognition [24].
In conclusion, our findings define a mechanism by which Pon1 prevents Aβ generation in a mouse model of AD and neural cells.
Author Contributions: Ł.W. performed and analyzed the experiments, carried out behavioral assessments; H.J. conceived the idea for the project, designed the study, generated Pon1 −/− 5xFAD mouse model, bred the mice, collected tissue samples, analyzed the data, and wrote the paper. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data that support the findings of this study are available in the methods and/or supplementary material of this article.