A Small Molecule-based Treatment Approach for Alzheimer’s Disease

Abstract

Alzheimer's disease (AD) is the most common form of dementia. The AD pathogenesis are hallmarked by the accumulation of Aβ, phosphorylated tau, and microglial-mediated neuroinflammation, which jointly leads to neuronal death and memory deficits. In the disease progression of AD, chronic Aβ and phosphorylated tau deposition trigger excessive activation of microglia and produce pro-inflammatory cytokines, thus triggering adjacent neuronal toxicity. Additionally, microglia manifest compromised phagocytotic ability in AD progression leading to insufficient phagocytotic clearance of the misfold toxic proteins, resulting in amyloid plaque (Aβ) deposition and further triggering Tau pathology. The toxicity of the Aβ and Tau pathology damages the neurons and synapses leading to failure of the neural circuit transduction thus inducing memory deficits. Therefore, inhibiting the accumulation of Aβ and phosphorylated Tau, suppressing microglial excessive activation while enhancing the microglial phagocytotic clearance of the Aβ and phosphorylated Tau might be able to rescue the disease progress of AD.

In this study, we aim to mine some small molecules in restraining the AD pathologies such as Aβ and phosphorylated tau deposition, neuroinflammation and rescuing memory deficits for therapeutic interventions of AD. Recent advances suggested that low-dose X-ray irradiation harbors neuroprotection effects by modulating the microglial function. However, the therapeutic dose of the X-ray is controversial. In the first part of study, we aimed to find out the optimal therapeutic dose of X-ray irradiation against AD. We found that low-dose X-ray irradiation (LDIR) at 400 mGy rescued the survival rate of the neuronal cell lines Neuron 2a under the challenge of Aβ1-42 peptide and completely reversed the memory deficits in the streptozocin-induced sporadic AD mouse model. LDIR at 400 mGy ameliorated the memory deficits in the 9-month-old 3xTg-AD mice (stands for the early stage of AD), while reducing both Aβ and Tau pathologies and facilitating microglial phagocytotic clearance of the Aβ plaques in the 17-month-old 3xTg-AD mice (stands for the advance stage of AD). We then harvested the hippocampus from the sham-irradiated/irradiated 3xTg-AD mouse at 9-month-old and 17-month-old and their age match wildtype mice to perform the bulk RNA sequencing (RNA-seq). We utilized the gene signature of the irradiated 3xTg-AD mouse to query the LINCS data for mining small molecules by bioinformatic analysis. We identify two small molecules, VUF6002 and dexamethasone, that might recapitulate the beneficial effect of LDIR in treating AD.

In the second part of study, we found that treatment with the VUF6002 at 2.5mg/kg completely reversed the memory deficits of the streptozocin-induced sporadic AD mice and 8-month-old APP/PS1 mice that develop amyloidosis in the brain. Chronic treatment of VUF6002 also showed a prominent effect in rescuing memory deficits, reducing the Aβ and phosphorylated Tau deposition, and reinforcing microglial phagocytotic degradation of Aβ in 14-month-old APP/PS1 mice and 17-month-old 3xTg-AD mice. VUF6002 is a histamine H4 receptors (Hrh4) antagonist while Hrh4 is expressed in neurons and microglia in CNS. Aiming to dissect the cell type-specific function of Hrh4 that contributes to AD pathologies, we employed genetic manipulation tools adeno-associated viruses (AAV), and lentivirus to specifically knockdown the gene expression of Hrh4 in neurons and microglia, respectively. We found that knockdown microglial Hrh4, but not the neuronal Hrh4, improved memory deficits in 8-month-old APP/PS1 mice. Additionally, knockdown microglial Hrh4 can inhibit gliosis, reduce Aβ and Tau pathology and improve memory deficits in 15-month-old 3xTg-AD mice. More importantly, VUF6002 treatment and knockdown of the microglial hrh4 in the 12-month-old APP/PS1 mice enhanced microglial phagocytotic ability towards Aβ in the flow cytometry experiments. These results indicated VUF6002 might target the microglial Hrh4 to enhance microglial phagocytotic clearance of Aβ and ameliorate the AD symptoms. VUF6002 is a non-FDA-approved drug, aiming to apply VUF6002 to clinical trials in the future, we validated the toxicity of the VUF6002 by applying 10 times the effective dose to normal male and female C57 mice. After 4 weeks of VUF6002 at 25 mg/kg treatment, mice well-tolerated VUF6002 and showed comparable body weight, food and water consumption, four limbs grip strength, and motor function to the vehicle-treated C57 male and female mice. VUF6002 did not induce liver or kidney injury revealed by whole blood and serum test, indicating VUF6002 might be a good candidate applying for clinical trials.

Dexamethasone was another candidate that might recapitulate the neuroprotection of LDIR. In the third part of study, we validated that chronic treatment of dexamethasone at 1mg/kg can rescue the memory deficit in 8-month-old APP/PS1 mice and 14-month-old APP/PS1 mice. Dexamethasone is an FDA-approved drug that can treat inflammation-related diseases including enteritis. Therefore, we speculated that dexamethasone might confer the anti-inflammation and neuroprotective effects by modulation of the gut microbiota composition. We harvested the fecal sample from the dexamethasone-/vehicle-treated 13-month-old APP/PS1 mice to perform the 16S RNA sequencing. We found that dexamethasone induced a drastic change of the microbial composition in the 13-month-old APP/PS1 mice. Targeted metabolites studies showed that dexamethasone can enhance the production of microbial-derived short-chain fatty acid which harbors neuroprotective effect against AD. We will perform the fecal microbiota transplantation (FMT) from the dexamethasone-treated 15-month-old APP/PS1 mice to the native 11-month-old APP/PS1 mice to confirm the mechanism underlining dexamethasone’s neuroprotection effect.

In the fourth part of the study, we first confirm that chronic treatment of the neuroprotective small molecule Lycium Barbarum Polysaccharide (LBP) can reverse cognitive function in 8-month-old APP/PS1 mice. The hippocampus from LBP-/vehicle-treated APP/PS1 mice and their age-matched wildtype mice were harvested to perform bulk RNA sequencing. Through bioinformatic analysis, the differential expressed genes that were significantly modulated by LBP in the APP/PS1 mice were used to query the LINCS database for drug mining. We mined a novel small molecule Tenidap, a cyclooxygenase inhibitor and Kir2.3 channel activator, which might recapitulate the beneficial effect of LBP in treating AD. One month of treatment with Tenidap at 10 mg/kg significantly improved the spatial memory of the 8-month-old APP/PS1 mice. We will use two familiar AD mouse models at the advanced stage (14-month-old APP/PS1 mice and 17-month-old 3xTg-AD mice) to validate the beneficial effect of Tenidap against AD in the future.

In summary, we have successfully mined three small molecules VUF6002, dexamethasone, and Tenidap, which significantly improved AD symptoms in sporadic and familial AD mouse models. Our data suggested that VUF6002 might target microglial Hrh4 to inhibit microglia-mediated neuroinflammation and enhance the microglial phagocytotic clearance of Aβ, thus improving memory deficits in AD mouse models. Dexamethasone might ameliorate the memory deficits in the young and aged APP/PS1 mice, such beneficial effects might be granted by modulating of gut microbiota. Tenidap might inhibit cyclooxygenase activity and act as a potential anti-inflammation drug in treating AD. In the past decades, we have put tremendous effort into developing the treatment against AD but witnessed continual failure in directly targeting Aβ and phosphorylated Tau therapies. Our data suggested that combining the biology and bioinformatic methods can broaden the current strategies in treating AD. We might mine more small molecule candidates with novel mechanisms, or repurpose FDA-approved drug which can bypass the preclinical test and directly enter the clinical trials, for the therapeutic intervention of AD.

Date of Award	25 Aug 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Chi Him Eddie MA (Supervisor)