Targeting excess ceramides as an Alzheimer's disease intervention

Neurodegenerative disorders such as Alzheimer's Disease (AD) are increasingly associated with irregular lipid accumulation. Dysfunction in the catabolism of sphingolipids leads to many neurodegenerative disorders and has recently gained interest in AD. Excess ceramide deposition has been observed in amyloid-beta (Aβ) plaques, plasma, and cerebrospinal fluid of AD patients. Ceramide-lowering strategies have been underexplored as a treatment for AD and may prove beneficial in mitigating the disease. We used both pharmaceutical and genetic approaches to target ceramide accumulation in the 5xFAD mouse model of AD. Myriocin, an inhibitor of ceramide de novo synthesis, was administered to mice fed either a normal chow diet or a pro-ceramide diet. As a genetic approach, we developed an inducible loss-of-ceramides model by overexpressing Asah1 in neurons, the gene encoding the ceramide degrading enzyme acid ceramidase. We assessed the loss of ceramides as a preventative disease treatment. We assessed Aβ plaques and the activation and quantity of glia by immunohistochemistry. Mitochondrial function was assessed using high resolution respirometry. Cognitive behavior was assessed using Barnes mazes and fear conditioning chambers. Pharmaceutical inhibition of ceramide synthesis and targeted neuronal ceramide reduction enhanced cognitive outcomes and mitigated AD pathology in 5xFAD mice. Myriocin-treated 5xFAD mice exhibited reduced plasma and hippocampal ceramides, smaller Aβ plaques, fewer glial cells, improved memory, and mitochondrial function. Early neuronal Asah1 overexpression rescued memory loss but did not alter plaque size. However, it eliminated gliosis, restoring glial morphology to wild-type patterns. Our findings highlight ceramides as a promising AD therapeutic strategy.

ii ABSTRACT Neurodegenerative disorders such as Alzheimer's Disease (AD) are increasingly associated with irregular lipid accumulation. Dysfunction in the catabolism of sphingolipids leads to many neurodegenerative disorders and has recently gained interest in AD. Excess ceramide deposition has been observed in amyloid-beta (Aβ) plaques, plasma, and cerebrospinal fluid of AD patients. Ceramide-lowering strategies have been underexplored as a treatment for AD and may prove beneficial in mitigating the disease. We used both pharmaceutical and genetic approaches to target ceramide accumulation in the 5xFAD mouse model of AD. Myriocin, an inhibitor of ceramide de novo synthesis, was administered to mice fed either a normal chow diet or a pro-ceramide diet. As a genetic approach, we developed an inducible loss-of-ceramides model by overexpressing Asah1 in neurons, the gene encoding the ceramide degrading enzyme acid ceramidase. We assessed the loss of ceramides as a preventative disease treatment. We assessed Aβ plaques and the activation and quantity of glia by immunohistochemistry. Mitochondrial function was assessed using high resolution respirometry. Cognitive behavior was assessed using Barnes mazes and fear conditioning chambers. Pharmaceutical inhibition of ceramide synthesis and targeted neuronal ceramide reduction enhanced cognitive outcomes and mitigated AD pathology in 5xFAD mice. Myriocin-treated 5xFAD mice exhibited reduced plasma and hippocampal ceramides, smaller Aβ plaques, fewer glial cells, improved memory, and mitochondrial function. Early neuronal Asah1 overexpression rescued memory loss but did not alter plaque size. However, it eliminated gliosis, restoring glial morphology to wild-type patterns. Our findings highlight ceramides as a promising AD therapeutic strategy. iii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 BACKGROUND 3 METHODS 9 RESULTS 18 DISCUSSION 28 REFERENCES 34 1 INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder affecting an estimated 5.5 million people in the United States, making it the sixth leading cause of death in the United States [1]-[2]. It is characterized by cognitive decline, memory loss, neurotoxic amyloid-beta (Aβ) plaques and neuronal atrophy [3]. While these pathological features are well established, new evidence suggests that lipid metabolism plays a role in the progression of AD [4]-[6]. Ceramides are bioactive lipids that serve as essential components of cellular membranes and are involved in diverse cellular processes, including signal transduction, apoptosis, and differentiation [7]-[8]. Ceramides have gained significant attention due to their contributions to oxidative stress, inflammation, and neuronal cell death [9]-[11]. Notably, elevated ceramide levels have been reported in both the brains and serum of AD patients, as well as in 5xFAD transgenic mice, a widely used model of AD [12]-[14]. Furthermore, ceramides have been implicated in insulin resistance—a metabolic impairment increasingly linked to AD and often referred to as type 3 diabetes [15]. Research from the Holland Lab has demonstrated that inhibition of ceramide synthesis can restore insulin sensitivity and reduce tissue pathology in models of metabolic dysfunction [16]. However, the mechanisms linking ceramide metabolism to AD remain unclear. Previous studies have failed to differentiate the role of ceramides in the various cell types of the brain. I hypothesize that decreased neuronal ceramides will attenuate AD progression by improving cognitive function, generating smaller Aβ neuroinflammation and improving mitochondrial metabolism. plaques, lowering 2 No studies have shown that targeting ceramides in neurons alters morphological changes or addresses inflammation. Our approach uses pharmaceutical and genetic approaches to target ceramide accumulation in the 5xFAD mouse model of AD. We aim to investigate the impact of neuronal ceramide levels on cognitive behavior, Aβ plaque formation, neuroinflammation and mitochondrial function in AD. This approach will provide novel insights into whether targeting ceramide metabolism could represent a viable therapeutic strategy for AD. 3 BACKGROUND AD pathology consists of three hallmark features: Aβ plaques, neurofibrillary tangles, and hyperphosphorylated tau protein. Despite the focus on these pathological features, there has been limited success in treating AD [17]. As a result, researchers have begun exploring other contributing mechanisms, particularly lipid metabolism [18]. Ceramides are bioactive lipids involved in numerous cellular processes including apoptosis and membrane signaling [9]-[11]. Elevated ceramide levels correlate with numerous diseases including cardiovascular, metabolic, and neurodegenerative [19]. Recent advancements in lipidomics (the characterization of lipids including their structures, functions, and how they interact with other molecules) have allowed for further analysis of ceramides. Mass spectrometry studies have revealed that ceramides are elevated in AD patients' brains and localized within Aβ plaques [20]-[21]. Furthermore, circulating ceramide levels are elevated in individuals at risk for AD, marking them as potential biomarkers for early diagnosis [22]. Ceramides serve as the fundamental structural units of complex sphingolipids and are tightly regulated by multiple biosynthetic and catabolic pathways (Figure 1). The de novo synthesis of ceramides begins in the endoplasmic reticulum with serine palmitoyltransferase (SPT) catalyzing the formation of sphinganine [23]. This enzyme condenses serine and palmitoyl-CoA to form 3-ketosphinganine, which is then quickly reduced to sphinganine. Notably, SPT can produce deoxysphingolipids when it incorporates alanine or glycine in place of serine, and these atypical lipids are neurotoxic and accumulate in disorders like hereditary sensory neuropathy [24]-[26]. SPT can also 4 Figure 1. De novo synthesis of ceramides. Ceramide biosynthesis begins with palmitoyl-CoA and serine, catalyzed by serine palmitoyltransferase (SPT). Ceramide synthases incorporate acyl chains (R), and dihydroceramide desaturase form the final double bond. Ceramides can be further metabolized into sphingomyelin or degraded into sphingosine and sphingosine-1-phosphate. Myriocin inhibits SPT. Ceramide and acid ceramidase (its degradative enzyme) are also highlighted in red. utilize alternative fatty acid substrates other than palmitoyl-CoA. Although few studies indicate biological roles for these less abundant sphingolipids, their functions remain largely unexplored [27]-[28]. Following sphinganine synthesis, ceramide synthases acylate the sphingoid base to produce dihydroceramides, using fatty acyl-CoAs ranging from 14 to 34 carbons in length [29]-[31]. These dihydroceramides are then converted into ceramides through the insertion of a double bond by dihydroceramides desaturases [32]. Ceramides can also be generated from sphingolipids including sphingosine and sphingomyelin 5 allowing cells to regulate ceramide levels in response to metabolic cues. In lysosomes, acid sphingomyelinase cleaves sphingomyelin to release ceramides, which may then be deacylated by acid ceramidase to produce sphingosine and a free fatty acid [33]-[34]. This sphingosine can be reacylated by ceramide synthases via the salvage pathway, restoring ceramide levels, or it can be phosphorylated to generate sphingosine-1-phosphate, which is ultimately degraded [35]. Collectively, this pathway generates a diverse array of ceramide species, each with distinct functional properties across tissues. To further investigate the relationship between ceramides and AD pathology, researchers have turned to animal models. The 5xFAD mouse is one of the most studied AD models and has been instrumental in enhancing the understanding of the biochemistry of AD. 5xFAD mice overexpress five humanized mutations associated with Familial Alzheimer's Disease (FAD) expressed under the Thy1 promoter [36]. These include three mutations in the amyloid precursor protein (APP): Swedish (K670N/M671L), Florida (I716V), and London (V717I) variants. In addition, it carries two mutations in Presenilin1 (PSEN1), M146L and L286V. This model reproduces key features of AD pathology including increased Aβ42, the insoluble and neurotoxic peptide associated with worse outcomes. They are a particularly attractive model because of the speed with which the mice begin to show extracellular Aβ accumulation. Aβ deposition and gliosis are measurable at two months of age. By six months of age, Aβ42 plaques are pronounced in males and females, and ceramide levels are higher in 5xFAD mice than wild-type controls [12]. This mouse model exhibits elevated ceramide levels in both the brain and serum [13][14]. Moreover, acid ceramidase (N-acylsphingosine amidohydrolase 1, encoded by Asah1) is responsible for the degradation of ceramides within the lysosome. Asah1 is 6 downregulated in neurons from AD patients [37] and is similarly downregulated in neurons of 5xFAD mice [38]. The accumulation of these lipids is significant because ceramides are known to play a role in AD pathology. The elevated ceramide levels in the 5xFAD model reflect a relationship between Aβ plaque deposition and disrupted lipid metabolism. Increased Aβ levels enhance sphingomyelinase activity, leading to higher ceramide abundance [11]. This is particularly concerning as excess ceramides can induce apoptosis in neurons grown in culture [7]. This neurotoxicity arises partly due to ceramides' capacity to disrupt mitochondrial function. Ceramide accumulation leads to the opening of the mitochondrial permeability transition pore (mPTP), which results in mitochondrial swelling, depolarization, and ultimately cell death [39]. This process allows intermembrane proteins, particularly cytochrome c, to escape and activate the intrinsic apoptotic pathway [40]. This disruption exacerbates oxidative stress by increasing the production of reactive oxygen species (ROS), which further damages mitochondrial DNA, proteins, and lipids, creating a self-perpetuating cycle of cellular dysfunction and neurodegeneration [41]. Beyond mitochondrial impairment, ceramides also exacerbate endoplasmic reticulum stress, another key pathological feature of AD. [42] Elevated ceramide levels can trigger the unfolded protein response (UPR), leading to the upregulation of pro-apoptotic mediators such as DNA damage-inducible transcript 3 (CHOP) and protein kinase R-like endoplasmic reticulum kinase (PERK), further sensitizing neurons to stress-induced apoptosis. Moreover, the dysregulation of ceramide metabolism has been implicated in tau pathology, as ceramides have been shown to facilitate tau hyperphosphorylation, thereby accelerating the formation of neurofibrillary tangles [43]. 7 Neuroinflammation is another critical factor linking ceramide accumulation to AD pathology. Ceramides play a pivotal role in activating inflammatory signaling pathways, particularly through their modulation of toll-like receptors (TLRs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. Elevated ceramide levels have been shown to enhance the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), which contribute to chronic neuroinflammation and exacerbate neuronal damage [44]. Additionally, ceramides promote the activation of microglia and astrocytes, leading to a sustained inflammatory response that further amplifies oxidative stress and synaptic dysfunction [45]-[46]. This suggests that ceramides act at multiple levels to promote neurodegeneration, making them a compelling target for therapeutic intervention in AD. Another source of ceramides come from the catabolism of sphingomyelin by neutral sphingomyelinase (nSMase2). In support of the hypothesis that regulating ceramides may improve AD pathologies, the systemic genetic ablation of nSMase2 lowers ceramides, depletes exosome trafficking, combats plaque formation, and improves cognition in 5xFAD mice [14]. This has provided proof-of-concept for the therapeutic potential of targeting ceramides. However, this ablation eliminates formation of exosomes, and it remains unclear if sphingolipids or the trafficked exosome cargo are the drivers of pathology. Moreover, sphingolipids play critical roles during neuronal development [47]. Genetic manipulation of ceramides must be restricted to adulthood to avoid developmental confounds. A major limitation of these previous studies is their lack of cellular specificity. Most approaches that modulate ceramide levels do so globally, affecting all cell types throughout 8 the body or brain. This has made it difficult to discern the specific contribution of ceramides in individual cell types, particularly neurons, which are most affected in AD. While many studies have shown an association of ceramides in both AD patients and rodent models, very few studies have probed the role of ceramides as a causative factor in AD. To our knowledge the aforementioned global knockout of neutral sphingomyelinase, which reduces exosome secretion globally, is the only genetic intervention which has evaluated a role for ceramide in AD. To test our hypothesis, we employ two complementary approaches. First, we will use myriocin to reduce systemic ceramide levels and determine its effect on AD progression. Myriocin is a potent inhibitor of SPT, the enzyme responsible for the ratelimiting step in ceramide biosynthesis [48]. Previous studies have validated myriocin's potential to traverse the blood-brain barrier (BBB) [49]. Given that over 98% of small molecule drugs struggle to cross the BBB [50], myriocin’s ability to do so positions it as a suitable candidate for assessing the impact of ceramide reduction on AD pathology. Although myriocin lacks cell-type specificity, it targets de novo synthesis of ceramide unlike the studies using nSMase whole-body knockout. In parallel, we will use a transgenic approach to selectively lower ceramide levels in forebrain neurons, testing whether neuronal ceramide specifically contributes to AD pathogenesis. 9 METHODS A. Pharmaceutical Myriocin Treatment All procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Utah (Protocol #00001986). Male 5xFAD mice from The Jackson Laboratory (JAX#00873) were crossed with female C57BL/6J mice (JAX#000664) to establish a colony. Myriocin was used to block ceramide de novo synthesis. Eight-week-old 5xFAD mice wild-type littermates were treated every other day with myriocin (0.3 mg/kg, intraperitoneal injection) while maintained on normal-chow diets until 25 weeks of age, at which time, these mice were subjected to a Barnes Maze to test cognitive function (see Section G for details). We additionally challenged 6-week-old 5xFAD mice and wild-type littermates with an obesogenic pro-ceramide high-fat (HFD, 60% kcal from fat) for 19 weeks to test if elevated ceramides worsened AD pathology [51][52]. At eight weeks of age, these mice began myriocin-treatment until euthanasia at 25 weeks of age. B. Genetic Mouse Model 5xFAD:Camk2α-AC 5xFAD-Camk2αCreERT2:Rosa26fl-STOP-fl-rtTA: TRE-ASAH1 mice allow ceramide-degrading acid ceramidase (AC) to be increased in a cell-specific doxycyclineinducible, and dose-dependent manner (Figure 2). The previously described 5xFAD mice were bred into an established colony of mice that express Camk2a-CreERT2 (from The Jackson Laboratory, JAX#012362), a loxp-STOP-loxp rtTA in the Rosa26 locus (JAX#00567) and a TRE-ASAH1 transgene generated by Holland at the University of 10 Texas Southwestern [53]. Here, ceramidase overexpressing neurons can degrade ceramides regardless of their origin (i.e., de novo synthesis in the neuron, or ceramides coming from other sources such as glia or sphingomyelinase). A tamoxifen-inducible Cre recombinase allows for the removal of a STOP cassette flanked by loxP sites followed by the gene for the Reverse Tet Transactivator (rtTA). At eight weeks of age, mice were administered tamoxifen (Sigma T5648, 200mg/kg/day for 5 consecutive days by oral gavage) to induce Cre-mediated recombination. The mice were then placed on a doxycycline diet (600 mg/kg chow) which induces expression of Asah1 under the control of a Tet Response Element (TRE). Mice were bred to achieve equal numbers of 4 key genetic groups: 1) Figure 2. Camk2αAC-Tg mice. Schematic representing the doxycycline inducible acid ceramidase mouse system. Cell specific Cre allows for the expression of rtTA, which in the presence of doxycycline binds to the TRE allowing for overexpression of acid ceramidase. Original figure created by Joesph L. Wilkerson, used with permission. 11 5xFAD:Camk2αAC-Tg (5xFAD mice with AC overexpression); 2) 5xFAD:Camk2αNoCre (5xFAD mice lacking the AC transgene); 3) Camk2αAC-Tg (mice with AC overexpression) and 4) Camk2αNoCre (wildtype or WT). C. Assessing Lipidomics Mice were assessed at week 30 (Figure 3). Plasma was taken directly after euthanasia. Brains were immediately dissected on ice into major regions [cortex (primary motor and somatosensory areas) and whole hippocampus]. Lipids were extracted and quantified by targeted LC-MS/MS mass spectrometry (with internal standards) by the University of Utah Mass Spectrometry and Proteomics Core using a broad approach that quantitates 361 diverse lipids, including: 80 sphingolipid species (ceramides, hexosylceramide derivatives, sphingosine, S1P, etc), acylcarnitines, cholesterol, cholesterol esters, phospholipids and lysophospholipids [54]-[55]. Lipids were analyzed by lipid ontology (LION) enrichment analysis [56]. Figure 3. Experimental Setup. Timeline illustrating the experimental design for studying lipidomics, behavior, and neuroinflammation in a cohort of mice aged for 31 weeks. At week 8, transgenic mice are administered tamoxifen and a doxycycline diet. At week 25, assessments include Barnes Mazes tests and metabolic cage analysis. Week 30 analyses include inflammation, lipidomics, and immunohistochemistry (focusing on plaque formation, gliosis, dendritic spine loss and neuron loss). 12 D. Tissue Preparation for Histology and Immunohistochemistry Mice were cardiac perfused with phosphate-buffered saline (PBS) for 2 minutes to clear all blood from the vascular system, followed by a 5-minute perfusion with 4% paraformaldehyde. The brain was removed and placed in 10% neutral buffered formalin for 48 hours at 4°C. After fixation the tissue was washed in PBS to remove residual formalin and infused with 25% sucrose as a cryoprotectant. Brain tissue was embedded in 1:1 OCT/TFM (optimal cutting temperature/tissue freezing media) and frozen in a bath of 2-methylbutane (-20°C). Cryomold blocks were stored at -80°C until processed. For the myriocin studies, brain samples were cut into 20 µm sections on a Leica Cryostat and placed on TrueBond positive-charged glass slides. These were allowed to dry to adhere fully to the slides and stored at -80°C until used for immunohistochemistry. For the genetic mouse lines, brains were cut into 70µm thick sections and placed in a 6-well tissue culture plate. These were stored at -80°C until stained as free-floating tissue sections for immunohistochemistry. Immunohistochemistry was performed by immersing the slides in ice-cold 50% methanol for 30 seconds as a secondary fixation step. The slides were then brought to room temperature and allowed to dry. A hydrophobic barrier was made using a PAP (PeroxidaseAntiperoxidase) pen around each section. Free-floating tissue sections were washed in PBS to clean away the tissue-cutting matrix. All sections were then blocked in 2% bovine serum albumin for 1 hour, and the primary antibody was applied, followed by a 12-hour incubation at 4°C. The slides were washed with PBS, then species-specific secondary antibodies were applied for 1 hour. The slides were again washed in PBS followed by 13 treatment with TrueBlack (Biotum) to quench autofluorescence. Cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). The slides were then mounted with coverslips using Prolong Glass mounting media (ThermoFisher). E. Quantifying Aβ plaque formation Aβ plaques were assessed at week 30 (Figure 3). Aβ content in different brain regions and plasma were measured by a human Aβ42 and Aβ40 multiplex kit (ThermoFisher) and read on a Luminex MagPix system. Aβ plaques were labeled by immunocytochemistry [α-AβPAN (Cell Signaling 42284, 1:100 dilution from stock), αAβ40 (Invitrogen 44136, 1:200 dilution from stock), or α-Aβ42 (AbCam 201060, 1:1000 dilution from stock) using frozen, fixed brain tissue. The size and number of plaques were quantified via Adobe Photoshop. F. Quantifying Dystrophic Neurites. Fixed, frozen tissue from 30-week-old mice was sectioned and probed for dystrophic neurites at Aβ plaques by staining with α-βIII Tubulin and α-VDAC (Invitrogen MA1-118 & PA1-954A, 1:100 dilution from stock) along with synaptic markers α-bassoon (Cell Signaling 6897, 1:100 dilution from stock) and α-synaptophysin (Millipore 5258, 1:100 dilution from stock) to assess neurite number, size, mitochondrial content, and synapse numbers (Figure 3). 14 G. Monitoring Gliosis and Neuroinflammation Astrocytes (α-GFAP, Abcam 4674, 1:200 dilution from stock) and microglia (αIBA1, Wako 019-19741, 1:200 dilution from stock) were assessed using immunohistochemistry using frozen, fixed brain tissue. Cell numbers, density, and arborization were quantified from the images to determine gliosis in major brain regions. Microglial activation was assessed using antibodies to HLA-DR, known to stain activated glia at Aβ plaques [57]. We evaluated IBA1 and HLA-DR with α-TREM119 (Invitrogen 119902, 1:100 dilution from stock), a marker specific to microglia but not other invading monocytes to differentiate the two cell populations. H. Assessing Cognitive Function Cognitive function was measured by Barnes mazes and fear conditioning cages at 25 weeks of age (Figure 3). The Barnes maze is a spatial learning and memory task designed to evaluate hippocampal-dependent navigation [58]. It consists of a circular platform with multiple evenly spaced holes around its perimeter, only one of which serves as an escape route leading to a concealed box. The task relies on an animal’s natural aversion to open spaces and its drive to seek shelter [59]. Behavioral performance was assessed by measuring latency, or the time taken to locate and enter the escape hole [60]. Video recordings of the trials were analyzed using Adobe Premiere Pro (Adobe Systems, San Jose, CA, USA) to track and quantify latency. For the fear conditioning task, associative learning and memory were evaluated in two phases: contextual fear conditioning and cued fear conditioning [61]. During training, animals were placed in a conditioning chamber and exposed to a neutral auditory cue (tone) 15 paired with an electric shock. In the contextual phase, memory performance was assessed by re-exposing animals to the same conditioning chamber without the auditory cue or shock [62]. Freezing behavior (defined as the absence of movement) was measured and normalized to baseline pre-tone freezing levels to evaluate memory retention. In the cued phase, animals were placed in a new environment and presented with the auditory cue alone. Freezing behavior during the tone was recorded to assess the strength of the conditioned response [63]. 5xFAD mice are prone to excess wheel running which could complicate the interpretation of behavioral test results [64]. To address potential hyperactivity and energy balance, we used our comprehensive lab animal monitoring system (CLAMS) prior to running behavioral tests [65]. These can measure x, y, and z-axis movement, allowing us to evaluate potential hyperactivity in parallel. We conducted the CLAMS at 25 weeks. I. High-Resolution Respirometry Mitochondrial function in the brain was evaluated using the Oroboros Oxygraph2k (Oroboros Instruments, Innsbruck, Austria). Immediately after tissue collection, whole brain samples were placed in ice-cold isolation/homogenization medium (Buffer A: 100 mM sucrose, 100 mM KCl, 50 mM Trizma hydrochloride, 1 mM KH2PO4, 0.1 mM EGTA, 0.2% BSA, pH 7.4). The tissue was minced with surgical scissors, replenished with fresh buffer A after 5 minutes, repeating this for a total of three repetitions. Following the final mincing, a two-minute rinse with 1ml Buffer A and protease (10.6U/mg, Sigma-Aldrich; Cat. No P5380) was performed twice. The minced tissue was then homogenized in 2 mL of Buffer A using a water-cooled glass homogenizer at 150 rpm for 10 minutes, with a 16 vertical plunge of 5 cm / 30 secs, with a momentarily pause at the bottom. The homogenate was placed into a total of four chilled microcentrifuge tubes and centrifuged at 700g at 4°C for 10 mins. Supernatant was then transferred to a new microcentrifuge tube and centrifuged again at 700g at 4°C for 10 mins. The final supernatant was centrifuged again at 10,000g at 4°C for 10 mins to pellet the mitochondria. Supernatant was carefully removed and mitochondria pellets from each tube were resuspended and combined in 1 ml of Buffer A and underwent a second high-speed centrifugation at 7,000g for 5 minutes. The supernatant was carefully removed, and the mitochondrial pellet was resuspended in a resuspension medium (Buffer B: 225 mM sucrose, 44 mM KH2PO4, 12.5 mM Mg acetate, 6 mM EDTA, pH 7.1). Prior to loading isolated mitochondria in the Oroboros, protein concentrations were obtained using the bicinchoninic acid (BCA) assay method (Pierce Biotechnology, Rockford, IL) to determine total mitochondria yield. A total of 100 μg of mitochondrial protein was loaded into the Oroboros in mitochondrial respiration medium (MiR05, 0.5 mM EGTA, 3 mM MgCl2, 60 mM K‐lactobionate, 20 mM Taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1 g/L bovine serum albumin, pH 7.1) for measuring H2O2 production and mitochondrial respiration. High-resolution respirometry was performed using a substrate-uncoupler-inhibitor-titration (SUIT) protocol. Prior to the protocol, superoxide dismutase, amplex red and horseradish peroxidase were added to the Oroboros to detect H2O2 emissions. The SUIT protocol utilized the following substrates:· glutamate (10 mM) and malate (2 mM) for Complex I activity, ADP (5,000 μM) for complex I oxidative phosphorylation, succinate (10 mM) to activate Complex II and measure oxidative phosphorylation, rotenone (0.5uM) to inhibit complex I, oligomycin 17 (5uM) to inhibit complex V to determine respiratory control ratio, and antimycin A (2.5uM) to inhibit complex III for background correction. J. Statistics Our experimental design and methods controlled for biological variables including gender (both sexes were assessed and analyzed independently) and background (littermate controls), doxycycline treatment of experimental groups and controls, random assignment of mice to different experimental groups, sample size to achieve at least 80% power to detect significant relevant differences, and blinding of all operators during data collection and histological scoring. Cohorts of N=12 will detect a difference of 20% in latency with an error level of 5% and error level of 80%. Normality of data was assessed using the Shapiro-Wilk test. ANOVA with appropriate corrections for post-hoc analyses was used for comparisons among parametric groups, while the Mann-Whitney U test was applied for non-parametric comparisons. 18 RESULTS A. Reduction of Ceramide Accumulation and Neuroinflammatory Markers in 5xFAD Mice Treated with Myriocin LC-MS/MS quantification of ceramide 18:0 concentrations (the most prominent ceramide found in the brain) had reductions in ceramide levels in the hippocampus of 5xFAD myriocin treated mice compared to 5xFAD vehicle controls, with levels decreasing from an average of 8663 pmol/mg to 7456 pmol/mg (p < 0.005). This significant reduction was also observed in the plasma, with ceramide levels decreasing from 8706 pmol/mg to 3538 pmol/mg (p < 0.0001). 5xFAD mice show significantly higher ceramide levels compared to their WT counterparts in the hippocampus and plasma (Figure 4). Figure 4. 25-week-old 5xFAD mice have increased ceramides in the hippocampus and plasma. Myriocin reduces ceramides in the 5xFAD mice. LC-MS/MS data for ceramide 18:0, the most prominent species in the brain, showing an increase in 5xFAD mice and their reduction in the hippocampus and plasma with myriocin treatment. Data is shown as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. 20 of cortex Aβ plaques was reduced from 196.5 to 114.3 (p < 0.05) as shown in Figure 6A. Median cerebrum plaque diameters were reduced from 16.14 µm to 14.18 µm (p < 0.0001) and median hippocampus plaque diameters were reduced from 17.67 µm to 14.07 µm (p < 0.05) as shown in Figure 6B. Myriocin's effect on microglia neuroinflammation was analyzed by evaluating IBA1-stained microglia. Figure 7A shows that myriocin treatment tends to reduce microglial in the cerebrum of 5xFAD mice, although the differences were not statistically significant. In the hippocampus, myriocin treatment significantly reduced microglial density in 5xFAD mice compared to vehicle-treated controls (p < 0.001, Figure 7B). Figure 6. 25-week-old 5xFAD mice fed HFD and treated with myriocin showed reduced Aβ plaques. A) Aβ plaque counts in the cortex. (B) Plaque size distribution in the cerebrum and hippocampus. Data are presented as mean ± SD. Figure A uses a two-way unpaired test (N=3). Figure B uses a Mann-Whitney U test (N=636,535,177,143 from left to right). *p<0.05. 21 Figure 7. 25-week-old 5xFAD mice fed HFD and treated with myriocin showed reduced microgliosis. A) Microglial counts in the cerebrum. B) Microglial counts in the hippocampus. Data are presented as mean ± SD. Figures A and B utilize a one-way ANOVA with Tukey's post hoc test (N=3). ****p<0.0001, ***p<0.001, ns = not significant. Furthermore, the number of cerebellum and hippocampus astrocytes were significantly reduced. Mean astrocytes numbers were reduced from 139.4 to 109.7 (p < 0.001) in the hippocampus and from 80.8 to 58.2 (p < 0.01) in the cerebrum (Figure 8A & 8B). As an indicator of astrocyte activation, we also counted arbors that extended from the cell body. 5xFAD myriocin-treatment reduced the number of arbors in the cerebrum, reducing the number of arbors per cell area from 5.35 to 4.28 (p<0.0001, Figure 8D). Astrocyte activation was unchanged in the hippocampus (Figure 8C). 22 Figure 8. 25-week-old AD mice fed HFD and treated with myriocin showed reduced astrogliosis. A) Astrocyte number in the hippocampus. B) Astrocyte counts in the cerebrum. C) Astrocyte arbor number in the hippocampus. D) Astrocyte arbor numbers in the cerebrum. Data are presented as mean ± SD. All figures utilize a one-way ANOVA with Tukey's post hoc test (N=3). ****p<0.0001, ***p<0.001,**p<0.01 ns = not significant. 23 B. 5xFAD Mitochondrial Function May Improve with Myriocin To investigate the effects of myriocin on metabolic function in 5xFAD mice, oxygen consumption rates were measured using high-resolution respirometry across different mitochondrial complexes. These included Complex I activity (pyruvate/malate), Complex I oxidative phosphorylation (ADP), oxidative phosphorylation from both Complex I and II (succinate), Complex II activity (rotenone), and Complex V inhibition (oligomycin). 5xFAD myriocin-treated mice had higher succinate levels than AD vehicle mice. Mean succinate activity was increased from 39.15 nmol/min/mg to 54.21 nmol/min/mg, indicating a trend toward enhanced Complex II activity (Figure 9, not significant). As Figure. 9. Succinate-driven mitochondrial respiration may increase in 5xFAD mice treated with myriocin. The graph displays respiration rates across distinct metabolic states. Succinate-linked activity is higher in myriocin-treated 5xFAD mice compared to vehicle controls, though this difference is not statistically significant. Statistical significance is denoted by * (p < 0.05) and **** (p < 0.0001). N ranges from 8 to 11 per group. 24 succinate is a critical substrate for ATP production, reducing ceramide levels could help improve mitochondrial efficiency. While there were significant differences between other transgenic mice variants in the succinate group, no other significant differences were observed between groups in other metabolic states. C. Cognitive Function Improved with Myriocin and Asah1 Overexpression CLAMS analysis at 25 weeks revealed no statistically significant differences in hyperactivity between 5xFAD mice and WT controls (Fig 10A). Measurements of ambulatory activity indicated comparable activity levels across groups, suggesting that hyperactivity did not confound subsequent behavioral assessments. In the Barnes Maze task (Figure 10B), male 5xFAD myriocin-treated mice had reduced latency times (time taken to locate and enter the escape hole, p < 0.05). This effect was not observed in females where mice showed a modest but non-significant reduction in latency. Similarly, in Figure 10C, male 5xFAD:Camk2αAC-Tg mice had significant reductions in latency times compared to 5xFAD:Camk2αNoCre controls (p < 0.05). While female 5xFAD:Camk2αAC-Tg mice also showed lower average latency than controls, the difference did not reach significance. D. Neuronal Asah1 Overexpression Eliminates Gliosis and Improves Memory The effects of acid ceramidase overexpression on neuroinflammatory markers were assessed by examining IBA1-stained microglia and GFAP-stained astrocytes in the cortex and hippocampus of 5xFAD mice. Activation and migration of IBA1⁺ microglia/monocytes and GFAP⁺ astrocytes were eliminated in 5xFAD:Camk2αAC-Tg mice. 25 Fig. 10. A) 25-week-old 5xFAD mice do not exhibit significant hyperactivity. Ambulatory activity was recorded over a 48-hour period using the Comprehensive Lab Animal Monitoring System (CLAMS) with white and gray backgrounds indicating 12-hour light and dark phases, respectively. B) Male myriocintreated 5xFAD mice show improved cognitive behavior. 25-week-old mice were assessed for cognitive function by Barnes maze. Male 5xFAD mice treated with myriocin showed significantly lowered latency times. However, this is not observed in females. C) 5xFAD Asah1 overexpressing mice show improved primary latency times. Male 5xFAD:Camk2αAC-Tg mice showed significantly reduced latency times compared to 5xFAD:Camk2αNoCre control. No significant differences were detected in females. Data are presented as mean ± SD with one-way ANOVA by sex. *p < 0.05, **p < 0.01. Sample sizes appear beneath each bar. Plaque size and number remained unchanged (Fig 11). These findings suggest that neuronal acid ceramidase dampens the inflammatory response by shifting glial cells toward a less reactive state via altered ceramide metabolism. The effects of acid ceramidase overexpression on memory performance were assessed by measuring freezing behavior with fear conditioning cages. Contextual conditioning represents the ability of the mice to associate the environment with an aversive stimulus, while freezing during the post-tone phase reflects the ability to associate a specific auditory cue with the aversive stimulus. Freezing behavior in contextual 26 Figure 11. Acid ceramidase overexpression in forebrain neurons prevents gliosis in 5xFAD mice. Immunofluorescence staining of perfusion-fixed cortex (left) and hippocampus (right) in 25-week-old 5xFAD mice, showing reduced IBA1+ microglia (green) and GFAP+ glia (red) in the presence of acid ceramidase overexpression selectivity in neurons (bottom). AB plaques are magenta. Plaque size and number remained unchanged compared to 5xFAD control. conditioning was reduced in 5xFAD:Camk2αNoCre mice compared to WT controls, while freezing behavior in 5xFAD:Camk2αAC-Tg mice was restored to levels comparable to WT controls (Figure 12A). In the post-tone phase, 5xFAD:Camk2αNoCre mice exhibited minimal freezing, consistent with impaired fear memory recall. In contrast, 5xFAD:Camk2AC-Tg mice demonstrated an increase in freezing behavior, indicating a partial recovery of cued fear memory (Figure 12B). These results suggest that neuronal Asah1 overexpression restores memory deficits in 5xFAD mice. 27 Figure 12. Freezing events in fear conditioning cages are improved to wild-type levels in 25-week-old 5xFAD overexpressing Asah1. A) Percentage of freezing behavior in the contextual phase of fear conditioning. B) Freezing behavior during the post-tone phase, normalized to pre-tone levels. Sample sizes were N=3 for all groups, except for 5xFAD: Camk2αNoCre (N=2). No statistically significant differences were observed among groups. 28 DISCUSSION This study aimed to investigate the role of ceramide metabolism in Alzheimer’s disease (AD) pathology and the therapeutic potential of reducing ceramides in neurons. By using both pharmaceutical and genetic approaches in 5xFAD mouse models, we demonstrated that reducing neuronal ceramide overaccumulation can impact key pathological and behavioral features of AD. These include amyloid-beta (Aβ) plaque formation, neuroinflammation, mitochondrial function and cognitive behavior. Consistent with prior reports [16],[67], myriocin treatment resulted in significantly lower levels of ceramides in the plasma (Fig. 4). Reducing ceramide accumulation by myriocin showed a decrease in plaque formation, decreased neuroinflammation, improved mitochondrial function and improved cognitive performance in 5xFAD mice (Fig. 5 to Fig. 10B). Genetic overexpression of Asah1, which promotes ceramide degradation in neurons, alleviated memory deficits and gliosis but it did not reduce Aβ plaque size or number (Fig. 11). This suggests that ceramide-mediated neuroinflammation may be more critical to neuronal health than plaque morphology. Furthermore, Asah1 overexpression showed improved cognitive behavior demonstrating the potential of ceramide-targeted strategies to address age-related deficits (Fig. 10C & 12). These findings support the hypothesis that ceramides exacerbate neuronal damage and neuroinflammation, reinforcing the need to explore ceramide-targeting strategies as potential therapeutic approach for AD. Previous studies have shown the involvement of ceramides in promoting proapoptotic pathways within astrocytes; the work by Wang et al indicates that ceramideenriched exosomes play a role in astrocyte apoptosis associated with Aβ plaque toxicity [68]. Our results extend this work by demonstrating that reduced ceramide levels via 29 myriocin led to diminished activation of astrocytes and glia cells in the 5xFAD model (Fig. 5 to Fig. 8). This reveals that targeting ceramide production can alleviate neuroinflammatory responses in the AD brain. Interesting, our genetic model show that neuronal overexpression of the ceramidedegrading enzyme restored glial morphology in AD mice to resemble wild-type patterns (Fig. 11). One possibility is that neuronal ceramide metabolism influences glial activation via exported ceramide-rich exosomes, which may produce inflammatory signals independent of direct Aβ processing. Prior global approaches, such as neutral sphingomyelinase deletion, reduced ceramides and AD pathology but impaired exosome formation, leaving unclear whether the observed benefits were due to reduced sphingolipids, altered exosome cargo, or the cell type of origin [16]. By selectively reducing ceramides in neurons, our findings clarify this ambiguity, demonstrating that neuronal ceramide depletion is sufficient to attenuate gliosis. While these findings highlight the role of ceramide in driving neuroinflammation, its relationship with Aβ pathology remains less clear. While previous studies suggested a connection between ceramide metabolism and the enzymatic processing of amyloid precursor protein (APP) [69]-[70], our study showed that genetically decreasing ceramide levels did not significantly alter Aβ plaque characteristics (Fig. 11). Notably, myriocin treatment reduced Aβ plaques (Fig. 5), whereas Asah1 overexpression did not change plaque morphology (Fig. 11). It is possible that Asah1 overexpression could lead to alterations in ceramide metabolism that do not directly influence APP processing pathways or that these changes operate through different cellular mechanisms [71]. 30 Our study further suggests a link between ceramide metabolism and mitochondrial function in AD pathology. Mitochondrial dysfunction is a well-documented feature of AD, where impairments in oxidative phosphorylation contribute to neuronal deficits and oxidative stress [72]. We observed that myriocin treatment led to a trend of increased succinate-driven respiration in 5xFAD mice, suggesting enhanced Complex I/II activity (Fig. 9). This finding aligns with prior research indicating that ceramides disrupt mitochondrial electron transport chain function [40]. Given that succinate is a key substrate for ATP production, our results imply that lowering ceramide levels may alleviate mitochondrial inefficiencies observed in AD. Interestingly, this effect was not broadly observed across all metabolic states, reinforcing the idea that ceramide’s impact on mitochondrial function might be Complex I/II specific. Our findings also revealed intriguing sex-specific differences in cognitive behavior. Male 5xFAD mice treated with myriocin demonstrated a significant reduction in primary latency times in the Barnes maze, indicating improved spatial learning and memory (Fig. 10B). In contrast, the female 5xFAD cohort did not exhibit a comparable improvement following treatment. Similarly, in the Asah1 overexpression model, male mice showed a significant enhancement in cognitive performance, whereas the improvement in females was more modest and did not reach statistical significance (Fig. 10C). In support of this, recent literature indicates significant sex-based variations in brain and plasma sphingolipid profiles. Blot et al. (2021) demonstrated region-specific differences in ceramide levels between sexes, reporting that total ceramide levels were substantially lower in the hippocampus yet higher in the cortex and cerebellum of female mice compared to males [66]. Together with our findings, this suggests that sex-specific differences in sphingolipid 31 metabolism may influence outcomes in AD. However, it is important to note that these effects also arise from a combination of factors including hormonal regulation and genetic variation. Nonetheless, our findings suggest that ceramides do not merely reduce pathology at a biochemical level but also translate into improvements in cognitive performance. Importantly, these cognitive benefits were observed in early intervention models indicating that targeting ceramide metabolism may be effective at multiple stages of disease progression. A major limitation of this study is that the intended sample size (N=12 per cohort) was not met, reducing the statistical power to detect significant differences in latency and other behavioral or histological outcomes. Our initial power calculations indicated that an N=12 per group would provide 80% power to detect a 20% difference with a significance level of 5%. However, due to technical constraints the actual sample size was lower than planned. This reduction in sample size increases the risk of Type II errors. Future work should achieve a fully powered experimental design by ensuring sufficient numbers of mice to accommodate potential losses. Although commonly used in AD research, the use of 5xFAD mice models is not fully representative of all aspects of human AD pathology. Notably, 5xFAD plaque formation begins at around 2 months, much earlier than the decades-long preclinical amyloid deposition in humans [12]. The model also lacks the genetic diversity of human AD, which involves environmental and lifestyle factors not reflected in 5xFAD mice. Additionally, the 5xFAD model does not replicate tau pathology, limiting its ability to capture the amyloid-tau relationship observed in humans. While our model does not explicitly assess tau aggregation, prior studies suggest that ceramide dysregulation 32 contributes to tau hyperphosphorylation and cytoskeletal disruption [43]. Future work should examine whether ceramide-targeting interventions influence tau pathology, as this could provide a more comprehensive understanding of ceramide’s role in AD. Another consideration is the broader role of ceramide metabolism in systemic physiology. While ceramide reduction in the brain may be beneficial in the context of AD, ceramides also play crucial roles in immune function, metabolism, and cell survival. Future studies should examine whether myriocin has unintended effects on peripheral tissues and whether other targeted approaches could mitigate these concerns. Given the promising results observed in this study, targeting ceramide metabolism offers a promising avenue for novel AD therapies. However, multiple challenges must be addressed to translate these findings into effective treatments. Key among these is the development of pharmacological agents that selectively modulate ceramide levels in the brain. Localized drug delivery strategies could help maximize therapeutic efficacy while mitigating systemic toxicity and allow bypass of the blood-brain barrier. These approaches may be particularly relevant given ceramides’ diverse roles in metabolism and immune function throughout the body. Beyond drug development, future studies should evaluate sex-specific differences in response to ceramide-targeted treatments to refine therapeutic strategies. Investigating how age and genetic backgrounds interact with ceramide metabolism could also help interventions. Future work should clarify how ceramide levels develop across distinct phases of AD pathology, enabling more precise determination of when ceramide-targeted interventions could yield the greatest therapeutic benefit. Ultimately, refining ceramidebased approaches for clinical application will require validation. 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