Antiallergic drug desloratadine as a selective antagonist of 5HT2A receptor ameliorates pathology of Alzheimer's disease model mice by improving microglial dysfunction

Alzheimer's disease (AD) is a progressively neurodegenerative disease characterized by cognitive deficits and alteration of personality and behavior. As yet, there is no efficient treatment for AD. 5HT2A receptor (5HT2AR) is a subtype of 5HT2 receptor belonging to the serotonin receptor family, and its antagonists have been clinically used as antipsychotics to relieve psychopathy. Here, we discovered that clinically first‐line antiallergic drug desloratadine (DLT) functioned as a selective antagonist of 5HT2AR and efficiently ameliorated pathology of APP/PS1 mice. The underlying mechanism has been intensively investigated by assay against APP/PS1 mice with selective 5HT2AR knockdown in the brain treated by adeno‐associated virus (AAV)‐ePHP‐si‐5HT2AR. DLT reduced amyloid plaque deposition by promoting microglial Aβ phagocytosis and degradation, and ameliorated innate immune response by polarizing microglia to an anti‐inflammatory phenotype. It stimulated autophagy process and repressed neuroinflammation through 5HT2AR/cAMP/PKA/CREB/Sirt1 pathway, and activated glucocorticoid receptor (GR) nuclear translocation to upregulate the transcriptions of phagocytic receptors TLR2 and TLR4 in response to microglial phagocytosis stimulation. Together, our work has highly supported that 5HT2AR antagonism might be a promising therapeutic strategy for AD and highlighted the potential of DLT in the treatment of this disease.


| INTRODUC TI ON
Alzheimer's disease (AD) is a progressively neurodegenerative disease characterized by deficit of cognition and alteration of personality and behavior. Despite the enormous efforts in fighting against AD during the last three decades, there has been yet no effective medication to treat this disease (Brambilla, 2017). The histopathology of AD is mostly defined by the accumulation of amyloid-β (Aβ) plaques and formation of neurofibrillary tangles (NFTs) in brains (Vijayraghavan et al., 2018). Extracellular amyloid plaques are mainly formed by Aβ aggregation, which is believed to be a key step in the pathogenesis of AD (Parhizkar et al., 2019). Data from preclinical and clinical studies have indicated that the unbalance between Aβ generation and clearance is tightly associated with AD pathogenesis, and the microglia-mediated neuroinflammation induced by Aβ directly causes neuronal damage including neuron and synapse loss, which is one of the leading causes of AD progression (Zhong et al., 2019).
Microglia as the main immune cells in the brain participate in normal function and defense of central nervous system. Similar to periphery macrophages, microglia respond to micro-environmental disturbance by drastically altering phenotypes and functions (Chen et al., 2014). In the pathogenesis of AD, microglia cluster around amyloid plaques and phagocytize the extracellular harmful proteins through phagocytic receptors (e.g., Toll-like receptors, scavenger receptors, and TREM2) and further degrade them through lysosomal-dependent ways including autophagy (Lee et al., 2018). At the same time, microglia provoke the conversion of a 'resting' type into an anti-inflammatory phenotype involving homeostasis, regeneration, and neuroprotection (Lee et al., 2018). However, once the phagocytosis and degradation function of microglia is destroyed, excessive endogenous toxic proteins transform microglia from anti-inflammatory phenotype to a pro-inflammatory phenotype that is associated with inflammation response, neuronal damage, and death (Currais et al., 2014). Thus, it is suggested that improving microglial dysfunction to prevent microglia-mediated inflammation should be a potential strategy for treating AD (Zhong et al., 2019).
Currently, several kinds of clinical drugs against AD are available (e.g., cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists), but they can only relieve the pathological symptoms of the disease. As approximately 30%-50% of AD patients are accompanied by neuropsychiatric symptoms including depression and agitation, some antipsychotics are also commonly used as concomitant drugs for AD patients (Wilkins & Sambamoorthi, 2011). Although it was concluded that continued long-term treatment with antipsychotics may reduce the rate of dementia in AD patients, the underlying mechanism is still unclear (Moraros et al., 2017).
5HT 2A receptor (5HT 2A R) is a subtype of 5HT 2 receptor belonging to the serotonin receptor family (Lippold & Dewey, 2017). As a type of G protein-coupled receptor (GPCR) primarily coupling to Gαq signal transduction pathway, 5HT 2A R expresses widely throughout the central nervous system and participates in varied brain functions, such as appetite control, thermoregulation, and sustained attention (Zhang & Stackman, 2015). Notably, 5HT 2A R also func- In the current work, we reported that clinically antiallergic drug desloratadine (DLT, Figure 1a)  (h) Times of platform crossing in probe trials (n = 8). (i) Changes in fEPSP slope were recorded following high frequency stimulation (4 × 100 Hz) in hippocampal DG region of APP/PS1 mice, and treatment of DLT or AAV-si-5HT 2A R effectively ameliorated LTP impairment in that region (brain slice, n = 2 brain slices per animal; animal, n = 2). (j) Golgi-Cox staining assay and (k) its quantification results indicated that treatment of DLT or AAV-si-5HT 2A R reversed the spine deficiency of hippocampal neuron in APP/PS1 mice. Scale bar: 50 µm, 10 µm, respectively (brain slice, n = 3; cell, n = 10). (l) Jess assay and ( Figure 1a).
In addition, results of calcium flow assays against primary neurons and microglia showed that DLT dose-dependently inhibited calcium flow in neurons and microglia by IC 50 at 9.177 μM and 0.3185 μM, respectively ( Figure S1A,B). Moreover, 5HT 2A R knockdown deprived DLT of its capability in regulating calcium flow in neurons and glia ( Figure S1A,B). Thus, all results verified that DLT repressed calcium flow inhibition by its antagonism against Finally, selectivity of DLT against 5HT 2A R over other 5HTR subtypes and monoamine receptors was evaluated as depicted in

| DLT treatment ameliorated pathological behavior of APP/PS1 mice by antagonizing 5HT 2A R
In an attempt to evaluate the capability of DLT in ameliorating memory and cognitive impairments of APP/PS1 mice, the models of new object recognition, Y-maze, and Morris water maze were applied (for each test, n = 8).
In addition, to verify that DLT ameliorated the pathological behavior of APP/PS1 mice by antagonizing 5HT 2A R, the assays were also performed against the APP/PS1 mice with selective 5HT 2A R knockdown in the brain by injecting AAV-ePHP-si-5HT 2A R.

| Y-maze test
This test was used to assess the spatial working memory mediated by hippocampus and prefrontal cortex. As indicated in Figure 1d

| Morris water maze test
This test was used to assess spatial learning and long-term memory of mice. As indicated in Figure  . All values were presented as mean ± SEM. For animal tissue assays, ### p < 0.001 compared with WT group by t test, *p < 0.05, **p < 0.01, ***p < 0.001 compared with APP/PS1 group by two-way ANOVA. For cell assays, *p < 0.05 compared with DMSO or si-Ctrl by one-way ANOVA Immunofluorescence assay and (i) its quantification results demonstrated that treatment of DLT and AAV-si-5HT 2A R suppressed the nuclear translocation rate of NF-κB in hippocampal microglia of APP/PS1 (brain slice, n = 4; cell, n = 12). Scale bar: 10 µm. (j) Flow cytometry was performed to detect the M1/M2 microglial polarization in the brain of APP/PS1, and NOS2-PE antibody and CD206-APC antibody were used to mark M1 and M2 microglia, respectively. The results of flow cytometry showed that treatment of DLT or AAV-si-5HT 2A R promoted the M1 to M2 microglial polarization in the brain of APP/PS1 mice (n = 2; sample size = 4). All values were presented as mean ± SEM. # p < 0.05, ## p < 0.01 compared with WT group by t test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with APP/PS1 group by two-way ANOVA 2.3.2 | DLT ameliorated synaptic integrity in APP/ PS1 mice by antagonizing 5HT 2A R In addition, Golgi-Cox staining assay was also performed to detect synapse integrity of hippocampal neurons of mice. As shown in Figure 1j,k, DLT or AAV-si-5HT 2A R treatment obviously reversed the spine deficiency of hippocampal neurons in APP/PS1 mice (F 3,27 = 89.82, p < 0.0001).
In addition, as shown in Figure 1l,m and Figure  Notably, no significant difference was observed in the amelioration of synaptic plasticity and integrity between APP/PS1 + AAV-si- Thus, all abovementioned results demonstrated that DLT treatment protected against the loss of synaptic plasticity and integrity in APP/PS1 mice by antagonizing 5HT 2A R.

| DLT promoted microglial phagocytosis of Aβ in the hippocampus of APP/PS1 mice through 5HT 2A R-mediated TLR2/4 upregulation
Considering that hippocampus as an essential brain region for declarative memory and cognition is tightly related to the onset and development of AD and CA1 region as the main area of hippocampal uptake of serotonin is much vulnerable to damage by inflammation and oxidative stress (Teixeira et al., 2018;, CA1 region should function potently in 5HT-mediated hippocampus function in AD progress. As such, the hippocampal CA1 region (Figure 2a) was here selected as the main area for investigating the mechanism underlying the regulation of 5HT 2A R

| DLT suppressed Aβ level independent of amyloidogenic pathway
Given that 5HT 2A R was ever reported to participate in the regulation of amyloid precursor protein (APP) ectodomain secretion and Aβ generation (Nitsch et al., 1996), we investigated whether DLT reduced Aβ level involving APP protein suppression by Jess assay. As indicated in Figure

| DLT promoted microglial Aβ clearance by stimulating autophagy
In addition, as shown in Figure  2.6.2 | DLT treatment repressed NLRP3 inflammasome in the hippocampus CA1 region of APP/ PS1 mice by antagonizing 5HT 2A R In addition, we also inspected the potential of DLT treatment in repressing NLRP3 inflammasome by immunofluorescence assay.
As indicated in Figure 4e It was noticed that microglia could not yet be specifically isolated from several CD11b-positive cell populations by magnetic activated cell sorting due to the high homology between microglia and macrophages/monocytes (Deininger et al., 2000) and microglia and macrophages share the same marker proteins including CD206 (Ji et al., 2018); there is currently no specific M2 polarized marker protein targeting only microglia and CD206 has been applied to label M2-type microglia. In our assay, CD206 was used to identify M2-type microglia from separated microglia by CD11b magnetic beads, which might be disturbed by infiltration of peripheral macrophages in the brain but will not affect our conclusion.
As shown in Figure  Collectively, all results demonstrated that DLT treatment suppressed neuroinflammation in the brains of APP/PS1 mice by antagonizing 5HT 2A R.

| DLT ameliorated AD-like pathology independent of targeting H1 receptor
Additionally, given that DLT was ever reported to be an antagonist of H1 receptor (H1R) (M. Chen et al., 2015), assays with H1R siRNA were also performed in primary microglia. As indicated in Figure S7A-G, DLT ameliorated AD-like pathology (phagocytosis, autophagy and inflammation. See details in Appendix S1) independent of H1R targeting.

| DLT treatment regulated autophagy and inflammation in APP/PS1 mice through 5HT 2A R/ cAMP/PKA/CREB/Sirt1 pathway
Sirt1 as a deacetylase plays a potent role in regulating inflammation, neuroprotection and autophagy (Donmez & Outeiro, 2013), and 5HT 2A R antagonism promotes cAMP accumulation leading to activation of PKA/CREB pathway (Nagatomo et al., 2004). In addition, cAMP-response element binding protein (CREB) as a transcriptional factor functions potently in mediating Sirt1 transcription and is critical for cognitive improvement (Fusco et al., 2016). With these facts, 2.8 | DLT-mediated 5HT 2A R/cAMP/PKA/CREB/GR signaling was responsible for microglial phagocytic receptor TLR2/4 regulation Given that glucocorticoid receptor (GR) as a nuclear transcription factor is activated by CREB and binds to a specific DNA sequence on target genes including TLR2/4 for regulating gene transcription (Novaes et al., 2017) and DLT treatment has been determined to promote microglial phagocytosis by upregulating TLR2/4, we next inspected whether CREB/GR signaling was involved in DLT-mediated TLR2/4 upregulation.

| DLT regulated GR by targeting 5HT 2A R
Immunofluorescence assay results (Figure 6a 2.8.2 | DLT upregulated TLR2/4 expression through activating GR Next, GR antagonist Mifeprex  was applied in the assay against BV2 cells, and the results (Figure 6c, Results of transactivation and mammalian one-hybrid assays demonstrated that co-treatment of 666-15 abolished the promotion of GR transactivation induced by the treatment of DLT or si-5HT 2A R in HEK-293 T cells, implying that the DLT-mediated GR activation was independent of the direct combination of GR with DLT (n = 3). All values were presented as the mean ± SEM. For animal tissue assays, ## p < 0.001 compared with WT group by t test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with APP/PS1 group mice by two-way ANOVA.
For cell assays, *p < 0.05, **p < 0.01, ***p < 0.001 compared with si-Ctrl by one-way ANOVA In the assay, the GFP-GR-U2OS cell line overexpressing GR was at first used for detecting GR nuclear translocation. As shown in Figure  Together, all results demonstrated that DLT-mediated 5HT 2A R/ cAMP/PKA/CREB/GR signaling was responsible for microglial phagocytic receptor TLR2/4 regulation.

| DISCUSS ION
5HT 2A R dysfunction links to a series of disorders, including schizophrenia, depression/anxiety, and drug addiction (Zhang & Stackman, 2015). Antagonists of 5HT 2A R (e.g., Pimavanserin; Clozapine (Joshi et al., 2017)) as antipsychotics have been clinically used to improve symptoms of depression or anxiety of psychiatric patients by regulating depressive release of dopamine (Landolt & Wehrle, 2009). Here, we determined that 5HT 2A R antagonism efficiently alleviated AD-like pathology in APP/PS1 mice, and the underlying mechanism has been intensively investigated. Our findings have strongly provided new evidence on the role of 5HT 2A R antagonism in microglia regulation. Moreover, DLT is currently a clinically antiallergic drug, and its obtained preclinical and clinical data including the efficient amelioration of AD-like pathology in the current work should no doubt provide valuable references for subsequent development of anti-AD drug based on this "old drug".
Given the crucial beneficial role of microglial phagocytosis in synapse regeneration and microenvironment restoration, targeting microglia has been accepted as a promising strategy for drug discovery against AD (Piirainen et al., 2017). TLR2/4 signaling participates in the process of microglial recognition and phagocytosis of neurotoxic Aβ deposits (Vijayraghavan et al., 2018), and upregulations of CD14 and TLR2/4 have been determined in the brains of AD patients (Cameron & Landreth, 2010). TLR2/4 activation promoted cell uptake of AD-associated amyloid peptide and reduced Aβ level (Fiebich et al., 2018), addressing the potency of In conclusion, we determined that DLT effectively improved cognitive impairment in APP/PS1 mice by improving microglial dysfunction. Briefly, DLT reduced amyloid plaque deposition by promoting microglial Aβ phagocytosis and degradation, and ame-

| Study design
The goals of the study were to evaluate the potential of 5-HT 2A R antagonist Desloratadine in the amelioration of AD-like pathology of APP/PS1 mice (functional behavior, synaptic integrity and plasticity, Aβ pathology, and neuroinflammation) and investigate the underlying mechanism by assay against APP/PS1 mice with 5-HT 2A R

| Statistical analysis
All cell experiments were performed in triplicate to obtain three independently data. Each study was completed with listed number of samples in figure legends. All data were presented as mean ± SEM, and statistical p < 0.05 was considered significantly. t Test was performed to analyze the significant difference between WT and APP/PS1. Two-way ANOVA with Fisher's LSD test was performed to analyze the significant differences among APP/PS1, APP/PS1 + DLT, APP/PS1 + AAV-si-5HT 2A R, and APP/ PS1 + AAV-si-5HT 2A R + DLT. For cell assay, one-way ANOVA with Dunnett's post-test was performed to analyze the significant difference between multiple treatments and the control. The data were analyzed for statistical significance using the graphing program GraphPad Prism 7.

ACK N OWLED G M ENTS
We thank Chuanfeng Tang, Yifan Wang, Yunfeng Hu, and Tong Xie for their technical supports in instrument operation and images quantification.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. such, have full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.