Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells

Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ1-40 and Aβ1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ1-40 and Aβ1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ1-42 had more adverse effects than Aβ1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ1-40 and Aβ1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is expected to affect 13 million individuals, mostly older adults, in the U.S. by 2050 [1]. The main pathological characteristics of AD are the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein. These alterations are associated with the loss of synapses, mitochondrial structural and functional abnormalities, oxidative stress, inflammation, and neuronal loss. A growing body of experimental and clinical studies demonstrate an associative link between AD and cardiac diseases such as heart failure, ischemic heart disease, and atrial fibrillation [2]. It has been proposed that cardiac dysfunction leads to cerebral hypoperfusion (hypoxia) associated with brain oxidative stress and acidosis that, in combination with Aβ aggregation, provoke neuronal degradation and progression of AD [3]. Oxidative stress can induce further production of Aβ and tau protein, another critical component involved in the pathogenesis of AD [4]. These studies suggest a feedback loop embedded in the crosstalk between oxidative stress and Aβ aggregation that stimulates the development and progression of AD. Although a causal role of cardiac dysfunction in AD progression can be explained, at least partially, by oxidative stress induced by decreased

Mitochondrial Permeability Transition Pore (mPTP) Opening
The swelling of mitochondria as an indicator of mPTP opening was determined in freshly isolated mitochondria (50 µg) by monitoring the decrease in light scattering at 525 nm in the presence or absence of Ca 2+ [28]. The swelling buffer contained 125 mM KCl, 20 mM Tris-base, 2 mM KH 2 PO 4 , 1 mM MgCl 2 , 1 µM EGTA, 5 mM α-ketoglutarate, 5 mM L-malate, pH 7.1. Mitochondrial swelling was measured by adding Ca 2+ to a total (accumulative) concentration of 100, 200, 300 µM with 5-min intervals at 37 • C using the Clariostar (BMG Labtech, Cary, NC, USA). The rates of swelling were calculated as decrements of absorbance values per minute (∆A 525 ·min −1 ·mg −1 ) and presented as a percentage of control.

H9c2 Cardiomyoblasts
H9c2 cardiomyoblasts were cultured according to the manufacturer's recommendations (ATCC). Briefly, the cells were cultured in DMEM based modified media (4 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate) supplemented with 10% fetal bovine serum and 1% antibiotic solution (Sigma-Aldrich) and maintained in 95% air and 5% CO 2 at 37 • C. Cells maintained within 80-90% confluence from passages 3-10 were used in experiments. Mitochondrial bioenergetics, metabolism, and morphology of H9c2 cells are similar to primary cardiomyocytes [29].

Mitochondrial CRC Assay
Freshly harvested and permeabilized cells were incubated at 37 • C in the 0.1 mL of the incubation buffer (200 mM sucrose, 10 mM Tris-MOPS, 5 mM α-ketoglutarate, 2 mM malate, 1 mM Pi, 10 µM EGTA-Tris, pH 7.4) containing 100 nM Calcium Green-5N. Calcium was added to increase matrix Ca 2+ load, and the fluorescence intensity was recorded by the CLARIOStar microplate reader (BMG Labtech).

Fluorescence Immunocytochemistry
Cells were fixed and permeabilized with ice-cold methanol for 5 min. Fixed cells were washed with PBS 3 times, then blocked with 2% BSA in PBS for 30 min. Antibodies against Aβ (ab11132), ATP5A (ab176569), and calreticulin (ab92516) were used as per the manufacturer's recommendation (Abcam, Waltham, MA, USA). The cells were incubated with the primary antibodies overnight at 4 • C, and then washed 3 times with PBS and incubated with Alexa Fluor 488 anti-mouse and/or Alexa Fluor 594 anti-rabbit secondary antibodies (Thermo Fisher, Waltham, MA, USA) for 1 h at room temperature. After incubation with secondary antibodies, the cells were washed 3 times with PBS, and 100 nM DAPI was added to visualize the nucleus. Images were captured by Olympus IX73 microscope with LUCPLFLN40X objective using Cellsense Dimension (Olympus, Center Valley, PA, USA) software. Image compositions were made using ImageJ.

Oligomerization and the Treatments of Aβ Peptides
The Aβ peptides Aβ 1-40 and Aβ 1-42 purchased from Sigma-Aldrich and Bon Opus Biosciences (Millburn, NJ, USA) were prepared as described previously [30]. First, lyophilized amyloid peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to 1 mM. After the peptides were completely monomerized, they were lyophilized and re-dissolved in DMSO to 10 mM, then were diluted to 1 mM by adding deionized water. The peptides were diluted in culture media to 0.2 mM and incubated at 4 • C for 48 h to form oligomers, which were then applied to cultured cells to a final concentration of 10 µM [31]. Low-binding surface plastic wares were used to prepare the amyloid peptides. To treat H9c2 cells, media was changed to serum-free media, Aβ peptides were added, and incubated 48-96 h. For primary HCAEC, Vascular Cell Basal Media supplemented with VEGF-based Endothelial Cell Growth Kit was used without modification. The HCAEC growth media was refreshed every 2 days in the presence or absence of Aβ peptides for 2-20 days.

Analysis of Cell Viability
Cell viability was determined by the AlamarBlue™ Cell Viability Assay Reagent (Thermo Fisher) as previously described [32].

Analysis of Cellular ATP, ∆Ψm, and mtROS
Cells were live stained with 5 µM ATP-Red (Sigma-Aldrich), 10 µM JC-1 (Thermo Fisher), and 2 µM MitoSOX (Thermo Fisher) for quantification of cellular ATP levels, ∆Ψ m , and mtROS production, respectively, following the manufacturers' recommendation (Thermo Fisher). The fluorescence intensity of the dyes was measured using the CLAR-IOStar microplate reader (BMG Labtech). Fluorescence signals of ATP-Red and MitoSOX were normalized to the fluorescence intensity (blue signal) of the nucleus (Hoechst). JC-1 signals were presented as the red to green fluorescence intensity ratio of the dye.

Apoptosis Assay
For analysis of activated caspases 3 and 7, live cells were stained with 2 µM CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher) in the presence of 50 nM Hoechst 33342 (Thermo Fisher) as per the manufacturer's recommendation. Fluorescence signals were measured using the CLARIOstar microplate reader (BMG Labtech). The caspase fluorescence intensity was normalized to the fluorescence intensity (blue signal) of the nucleus (Hoechst).

Statistical Analysis
Data were analyzed using Student's t-test. Results are presented as mean ± SEM. p < 0.05 was considered statistically significant. The number of biological samples but not technical replicates were used as a sample size.
CLARIOStar microplate reader (BMG Labtech). Fluorescence signals of ATP-Red and Mi-toSOX were normalized to the fluorescence intensity (blue signal) of the nucleus (Hoechst). JC-1 signals were presented as the red to green fluorescence intensity ratio of the dye.

Apoptosis Assay
For analysis of activated caspases 3 and 7, live cells were stained with 2 μM CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher) in the presence of 50 nM Hoechst 33342 (Thermo Fisher) as per the manufacturer's recommendation. Fluorescence signals were measured using the CLARIOstar microplate reader (BMG Labtech). The caspase fluorescence intensity was normalized to the fluorescence intensity (blue signal) of the nucleus (Hoechst).

Statistical Analysis
Data were analyzed using Student's t-test. Results are presented as mean ± SEM. p < 0.05 was considered statistically significant. The number of biological samples but not technical replicates were used as a sample size.

Aβ Decreased Cell Viability and Impaired Cell Morphology
The cell viability of HCAEC grown in the culture media containing 10 μM Aβ1-40 or Aβ1-42 for 20 days was significantly decreased in comparison with control cells. The cell viability of Aβ1-40 or Aβ1-42 groups were 8.6% and 24% lower (p < 0.05) than the control, respectively ( Figure 1A). Morphological analysis using phase-contrast microscopy in Aβ1-42-treated cells revealed irregular-shaped lumps appearing bright compared to the control and Aβ1-40-treated cells ( Figure 1B). Incubation of H9c2 cardiomyocytes with 10 μM Aβ1-42 for 96 h resulted in a 39% (p < 0.05) decrease of cell viability, whereas Aβ1-40 reduced the cell viability only by 8% ( Figure 1C). H9c2 cells challenged with Aβ1-42 showed abnormally shrinking cell morphology, in addition to abnormal bright aggregates ( Figure 1D).

Aggregated Aβ Accumulated Inside of Cells
To investigate the possible inclusion of Aβ aggregates in subcellular compartments, primary HCAEC and H9c2 cardiomyocytes incubated with Aβ 1-40 or Aβ 1-42 were visualized using immunocytochemistry. Control primary HCAEC showed only low perinuclear signal, which could indicate a low level of Aβ and/or background staining, whereas Aβ 1-40 -treated cells demonstrated the higher intensity of the cytoplasmic signal. Primary HCAEC challenged with Aβ 1-42 showed a strong signal of irregular-shaped aggregates with a size of 1-50 µm (Figure 2A). Similar patterns of intracellular aggregation of Aβ were observed in H9c2 cells. The control group showed low perinuclear signal, whereas cells grown with Aβ 1-40 showed more cytoplasmic signal (in addition to the nuclei) compared to the control. Cells grown with Aβ 1-42 showed irregular-shaped aggregates in the cytoplasm ( Figure 2B) that were absent in the control.

Cells and Mitochondria Demonstrated Early Response to Aβ
To elucidate the possible cause of the Aβ-induced cell death, we investigated the earlier changes that could predispose the cells and mitochondria to further functional alterations leading to cell death. Incubation of primary HCAEC with 10 µM Aβ 1-40 or Aβ 1-42 for 48 h did not decrease the cell viability compared to the control ( Figure 4A). Analysis of cellular morphology revealed that Aβ 1-42 increased dark, grainy aggregates in the cell cytoplasm that were not observed in the control and Aβ 1-40 -treated groups ( Figure 4B). Aβ 1-42 did not affect mtROS levels after 48 h, but it increased cellular ROS by 59% (p < 0.05) ( Figure 4C,D). In addition, mitochondria were found depolarized in the cells incubated with Aβ 1-40 or Aβ 1-42 that demonstrated 27% (p < 0.05) and 45% (p < 0.05) fewer ∆Ψ m for Aβ 1-40 and Aβ 1-42 , respectively, in comparison with the control ( Figure 4E). No significant increase in caspase activation was observed in the presence of the Aβ ( Figure 4F). Aβ 1-42 increased ATP levels by 59% (p < 0.05) while Aβ 1-40 did not affect ATP ( Figure 4G). Likewise, the H9c2 cells incubated with 10 µM Aβ 1-40 or Aβ 1-42 for 48 h did not show any change in cell viability ( Figure 4H).

Cells and Mitochondria Demonstrated Early Response to Aβ
To elucidate the possible cause of the Aβ-induced cell death, we investigated the earlier changes that could predispose the cells and mitochondria to further functional alterations leading to cell death. Incubation of primary HCAEC with 10 μM Aβ1-40 or Aβ1-42 for 48 h did not decrease the cell viability compared to the control ( Figure 4A). Analysis of cellular morphology revealed that Aβ1-42 increased dark, grainy aggregates in the cell cytoplasm that were not observed in the control and Aβ1-40-treated groups ( Figure 4B). Aβ1- 42 did not affect mtROS levels after 48 h, but it increased cellular ROS by 59% (p < 0.05) ( Figure 4C,D). In addition, mitochondria were found depolarized in the cells incubated with Aβ1-40 or Aβ1-42 that demonstrated 27% (p < 0.05) and 45% (p < 0.05) fewer ΔΨm for Aβ1-40 and Aβ1-42, respectively, in comparison with the control ( Figure 4E). No significant increase in caspase activation was observed in the presence of the Aβ ( Figure 4F). Aβ1-42 increased ATP levels by 59% (p < 0.05) while Aβ1-40 did not affect ATP ( Figure 4G). Likewise, the H9c2 cells incubated with 10 μM Aβ1-40 or Aβ1-42 for 48 h did not show any change in cell viability ( Figure 4H).  Cardiomyocytes also showed dark, grainy aggregates in the cytoplasm of incubated with Aβ 1-42 for 48 h. The control and Aβ 1-40 -treated groups did not show remarkable changes in morphology ( Figure 4I). Aβ 1-42 induced a 17% (p < 0.05) increase of mtROS and a 19% (p > 0.05) increase of cellular ROS after 48 h of incubation ( Figure 4J,K). Aβ 1-42 induced a 15% (p < 0.05) decrease of the ∆Ψm ( Figure 4L) and increased caspase 3/7 activation by 15% (p < 0.05) compared with the control ( Figure 4M). Both Aβ 1-40 and Aβ 1-42 had no significant effect on the ATP levels in H9c2 cells ( Figure 4N). Analysis of mitochondrial CRC demonstrated high sensitivity of the cells to Aβ 1-42 . Incubation of primary HCAEC with 10 µM Aβ 1-42 for 48 h decreased the mitochondrial CRC by 15% (p < 0.05) compared to control cells ( Figure 5A,B). Likewise, 10 µM Aβ 1-42 induced a 20% (p < 0.05) decrease of the mitochondrial CRC in H9c2 cardioblasts ( Figure 5C,D). Aβ 1-40 did not affect the mitochondrial CRC in both types of cells. Cardiomyocytes also showed dark, grainy aggregates in the cytoplasm of incubated with Aβ1-42 for 48 h. The control and Aβ1-40-treated groups did not show remarkable changes in morphology ( Figure 4I). Aβ1-42 induced a 17% (p < 0.05) increase of mtROS and a 19% (p > 0.05) increase of cellular ROS after 48 h of incubation ( Figure 4J,K). Aβ1-42 induced a 15% (p < 0.05) decrease of the ΔΨm ( Figure 4L) and increased caspase 3/7 activation by 15% (p < 0.05) compared with the control (Figure 4M). Both Aβ1-40 and Aβ1-42 had no significant effect on the ATP levels in H9c2 cells ( Figure 4N). Analysis of mitochondrial CRC demonstrated high sensitivity of the cells to Aβ1-42. Incubation of primary HCAEC with 10 μM Aβ1-42 for 48 h decreased the mitochondrial CRC by 15% (p < 0.05) compared to control cells ( Figure 5A,B). Likewise, 10 μM Aβ1-42 induced a 20% (p < 0.05) decrease of the mitochondrial CRC in H9c2 cardioblasts ( Figure 5C,D). Aβ1-40 did not affect the mitochondrial CRC in both types of cells.  Immunocytochemical analysis of the primary HCAEC incubated with 10 µM Aβ 1-42 for 48 h showed aggregated Aβ throughout the cytoplasm ( Figure 6A, green). Mitochondria in Aβ 1-42 -treated cells showed mostly fragmented mitochondria, whereas control cells and the cells incubated with Aβ 1-40 presented a well-organized mitochondrial network ( Figure 6A, white). Likewise, mitochondria were more fragmented in H9c2 cardioblasts incubated with 10 µM Aβ 1-42 for 48 h (Figure 6B, white) and showed aggregated Aβ in the cytoplasm ( Figure 6B, green). Immunocytochemical analysis of the primary HCAEC incubated with 10 μM Aβ1-42 for 48 h showed aggregated Aβ throughout the cytoplasm ( Figure 6A, green). Mitochondria in Aβ1-42-treated cells showed mostly fragmented mitochondria, whereas control cells and the cells incubated with Aβ1-40 presented a well-organized mitochondrial network ( Figure 6A, white). Likewise, mitochondria were more fragmented in H9c2 cardioblasts incubated with 10 μM Aβ1-42 for 48 h (Figure 6B, white) and showed aggregated Aβ in the cytoplasm ( Figure 6B, green).

Aβ induced Dysfunction of Cardiac Mitochondria In Vitro
To investigate the possible direct effects of Aβ on the mitochondria, we measured mitochondrial swelling rates, mtROS, ΔΨm, and ATP levels in mitochondria that were isolated from healthy rat hearts. The mitochondria were incubated with 10 μM Aβ1-40 or Aβ1-42 for 10 min. Results demonstrated that Aβ1-40 and Aβ1-42 further increased the Ca 2+induced mitochondrial swelling rate by 34% (p < 0.05) and 97% (p < 0.05), respectively, in comparison with the control (Figure 7A,B). The swelling of mitochondria apparently was induced by mPTP opening since the addition of sanglifehrin A, a cyclophilin D inhibitor, completely prevented the mitochondrial swelling in control and Aβ-treated groups (Figure 7B). Aβ1-42 slightly (5%, p < 0.05) increased mtROS ( Figure 7C), decreased the ΔΨm by 12% (p < 0.05, Figure 7D), and increased ATP levels (8%, p < 0.05), ( Figure 7E). Aβ1-40 did not induce any remarkable changes in mtROS, ΔΨm, and ATP levels.

Discussion
Results of the present study demonstrate the direct detrimental effects of Aβ, particularly Aβ 1-42 , on cardiomyocytes and primary coronary endothelial cells. On the other hand, the cardiac cells demonstrated a different sensitivity to Aβ. Comparative analysis of early (48 h) responses for both cells revealed severe effects of Aβ 1-42 on the mitochondria. We have previously shown that the mitochondrial bioenergetics, metabolism, function, and morphology of H9c2 are similar to primary cardiomyocytes [29].
Several metabolic alterations could be involved in the adverse action of Aβ 1-42 on mitochondria. Aβ oligomers have been shown to disrupt cell membrane permeability and calcium homeostasis in neurons [33], cerebral endothelial cells [21,24,34], and cardiomyocytes [5]. Our results showed that Aβ decreased the cell viability of cardiac cells as well as primary HCAEC. Our study and others have reported that mitochondrial dysfunction and mitochondria-mediated apoptosis play a crucial role in the pathogenesis of both AD and heart failure [19,35]. However, despite these findings, it is not clear whether these changes are the cause or just another result of aging in AD and heart failure patients. Our study showed early signs of cellular and mitochondrial dysfunctions because of Aβ. In addition to cerebral vasculature, Aβ peptides were found in atherosclerotic lesions and platelets [36]. High blood levels of Aβ 1-40 in patients with coronary heart disease were identified as a marker that could predict a high risk of mortality [37]. Analysis of the hearts and brains of patients with AD demonstrated that the hearts of several patients contain Aβ deposits (Aβ 1-40 and Aβ  ) that are structurally similar to those found in the brain. Similar amounts of Aβ 1-40 and Aβ 1-42 peptides were also found in the hearts and brains of AD patients. [6]. The patients with Aβ deposition in the heart presented diastolic dysfunction, although none of them had a history of coronary heart diseases. Aβ induced decreased complex activity, H 2 O 2 production, ATP synthesis, state 3 and 4 respiration, and release of cytochrome c in rat muscle mitochondria [38]. Interestingly, similar mitochondrial dysfunction and apoptotic mechanisms were shown by our group in cerebral microvascular endothelial cells challenged with multiple Aβ variants, including Aβ 1-40 and Aβ 1-42 [19,21,24,26].
Our results demonstrated that Aβ increases mitochondrial or cellular ROS production, which, in turn, can induce further propagate Aβ and tau protein production [4]. Analysis of the hearts of AD patients resulted in the discovery of the presence of amyloid aggregates (Aβ 1-40 and Aβ 1-42 ) in cardiomyocytes and interstitial spaces, and that was associated with myocardial diastolic dysfunction [6]. Although it is not yet clear what is the source of the Aβ aggregates in the hearts of AD patients, accumulation of Aβ has been observed not only in the heart but also in other organs of AD patients [39], suggesting that circulating Aβ can be deposited or that these peptides can be produced in the heart, among other organs. Furthermore, circulating Aβ oligomers, recognized as the major toxic species for multiple cell types, could participate in the induction of cardiac or vascular endothelial dysfunction through oxidative stress mechanisms. On the other hand, amylin and amyloid deposits were found in the hearts of non-AD patients with diabetic and idiopathic cardiomyopathy [5,40].
Our data demonstrate that Aβ has a direct effect on isolated mitochondria, although Aβ is not produced in mitochondria [41]. Existing data on the transportation/localization of Aβ in mitochondria are still controversial. Accumulation of amyloid precursor protein across mitochondrial import channels (TOM and TIM) was detected in brain mitochondria of AD patients [42]. Alternatively, extracellular oligomeric Aβ aggregates may affect mitochondrial function by triggering cell membrane receptors (e.g., death receptors) and/or other signal transduction pathways that converge on the mitochondria [19]. Aβ has been shown by many studies, including ours, to diminish mitochondrial respiration and increase the levels of mtROS in multiple cell types, including neuronal and cerebral endothelial cells [19,21,24,25,34,43], and thus, can induce further Aβ and tau protein production [4]. However, there are few if any studies that investigate the direct effects of Aβ on cardiomyocytes and coronary endothelial cells.
Aβ neurotoxicity is associated with intraneuronal Ca 2+ dyshomeostasis; increased cytosolic Ca 2+ levels were detected in AD mice [44] and after application of soluble Aβ oligomers to the brain of wild-type mice [45]. Our results demonstrated that Aβ accelerated the mitochondria swelling caused by Ca 2+ overload, an indicator of the mPTP opening. Studies on the brain tissue and neuronal cells revealed that the mPTP opening induced by high Ca 2+ is one of the mechanisms that mediate the effects of Aβ and tau protein leading to mitochondrial dysfunction and cell death [46]. Our results showed that the cyclophilin D inhibitor sanglifehrin A completely inhibited mitochondrial swelling induced by Ca 2+ and by Ca 2+ + Aβ, suggesting the effect of Aβ on the swelling depends on the mPTP opening. Cyclophilin D, a major mPTP regulator in the matrix, was found increased in ADaffected brain regions; Aβ-cyclophilin D complex was detected in Aβ-rich mitochondria from AD brain and transgenic AD mice, suggesting that the effects of Aβ to induce mPTP opening are mediated through its interaction with cyclophilin D [47]. Conversely, genetic or pharmacological inhibition of cyclophilin D prevented Aβ-induced mPTP opening and cell death [48], decreased mitochondrial and neuronal perturbations, and improved learning and memory in AD [49].
Mitochondrial quality control mechanisms, including mitophagy and mitochondrial biogenesis and dynamics, are compromised by aging. A large number of factors and mechanisms maintain the structural and functional integrity of mitochondria, and modulation of their intensity/efficiency with aging apparently impairs structural and functional integrity of mitochondria and diminishes the mitochondrial quality control leading to mitochondrial dysfunction and, eventually, age-related diseases such as AD [50]. In this context, our results demonstrated that, among other factors, Aβ accumulation in cardiac cells with aging might play a certain role in mitochondrial/cellular dysfunction in the elderly population.

Conclusions
This study reveals adverse effects of Aβ, particularly Aβ 1-42 , on cardiomyocytes and coronary endothelial cells that could be mediated, among other mechanisms, through functional and metabolic alterations of mitochondria. Like neurons and cerebral endothelial cells, mitochondrial abnormalities might stimulate the intrinsic apoptotic pathway leading to cell death. Although the source of Aβ and mechanisms of accumulation of Aβ aggregates in the heart in the aged population and AD patients remains undiscovered, this study opens new perspectives for elucidating the potential role of Aβ in cardiac dysfunction. Funding: This study was supported by the grants from the National Institutes of Health (SC1GM128210 and R25GM061838 to SaJ; R01NS104127 and R01AG062572 to SF), the National Science Foundation (2006477 to SaJ), and the Pennsylvania Department of Heath Collaborative Research on Alzheimer's Disease (PA Cure, to SF).

Institutional Review Board Statement:
The animal study protocol was approved by the UPR Medical Sciences Campus Institutional Animal Care and Use Committee (protocol A762020117, approved on 11 February 2021).