Seung Hyun Baek

Inhibition of Drp1 Ameliorates Synaptic Depression, Aβ Deposition, and Cognitive Impairment in an Alzheimer's Disease Model

Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimers disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-β (Aβ) in neurons and neuropathology and cognitive functions in Aβ precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aβ-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aβ deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aβ-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD. SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimers disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-β (Aβ)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aβ deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.

Notch1 deficiency decreases hepatic lipid accumulation by induction of fatty acid oxidation

Notch signaling pathways modulate various cellular processes, including cell proliferation, differentiation, adhesion, and communication. Recent studies have demonstrated that Notch1 signaling also regulates hepatic glucose production and lipid synthesis. However, the effect of Notch1 signaling on hepatic lipid oxidation has not yet been directly investigated. To define the function of Notch1 signaling in hepatic lipid metabolism, wild type mice and Notch1 deficient antisense transgenic (NAS) mice were fed a high-fat diet. High-fat diet -fed NAS mice exhibited a marked reduction in hepatic triacylglycerol accumulation compared with wild type obese mice. The improved fatty liver was associated with an increased expression of hepatic genes involved in fatty acid oxidation. However, lipogenic genes were not differentially expressed in the NAS liver, suggesting lipolytic-specific regulatory effects by Notch1 signaling. Expression of fatty acid oxidative genes and the rate of fatty acid oxidation were also increased by inhibition of Notch1 signaling in HepG2 cells. In addition, similar regulatory effects on lipid accumulation were observed in adipocytes. Taken together, these data show that inhibition of Notch1 signaling can regulate the expression of fatty acid oxidation genes and may provide therapeutic strategies in obesity-induced hepatic steatosis.

THE DRUG TG REDUCING BACE1 EXPRESSION LEVEL AND PREVENTING COGNITIVE IMPAIRMENT IN ALZHEIMER'S DISEASE MICE

used for chronic efficacy studies. Results: CNP520 is selective for BACE-1 over BACE-2 and highly selective over pepsin, cathepsin D & E, and renin. Low nanomolar inhibition of Ab and sAPPb release was observed in cell assays using wt-APP cells. The free fraction of CNP520 in the rat brain, and the concentration of CNP520 in the CSF, was comparable to unbound blood concentrations, indicating excellent brain penetration. Oral dosing of CNP520 reduced Ab in the rat brain by more than 80%. A single CNP520 dose in dogs reduced CSF Ab for 72 hours, in agreement with long terminal half-lives (9.5-23 hours) in animals. CNP520 did not induce any hair depigmentation when dosed to mice for 8 weeks at a dose for > 90% Ab reduction. No hypopigmentation was observed in chronic studies in transgenic mice, and during long-term toxicology studies. CNP520 was dosed into APP23 mice 6 months and showed dose-dependent reduction of Triton TX-100 soluble and insoluble Ab. At the high dose, the levels of deposited Ab40/42 were indistinguishable from baseline. Conclusions: Preclinical data predict that more than 80% Ab reduction can be reached in humans at steady state. CNP520 stopped further amyloid-b deposition in APP transgenic mice, indicating that the compound may be able to show long term efficacy against Ab deposition in humans.

Inhibition of Notch1 induces population and suppressive activity of regulatory T cell in inflammatory arthritis

Inhibition of Notch signalling has shown anti-inflammatory properties in vivo and in vitro models of rheumatoid arthritis (RA). The objective of this study was to determine whether Notch1 might play a role in regulating T-regulatory cells (Tregs) in animal models of RA. Methods: Collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) were induced in C57BL/6, Notch1 antisense transgenic (NAS) or DBA1/J mice. We examined whether pharmacological inhibitors of γ-secretase (an enzyme required for Notch1 activation) and antisense-mediated knockdown of Notch1 could attenuate the severity of inflammatory arthritis in CIA and CAIA mice. Proportions of CD4+CD25+Foxp3+ Treg cells were measured by flow cytometry. To assess the suppressive capacity of Treg toward responder cells, CFSE-based suppression assay of Treg was performed. Results: γ-secretase inhibitors and antisense-mediated knockdown of Notch1 reduced the severity of inflammatory arthritis in both CIA and CAIA mice. Pharmacological and genetic inhibition of Notch1 signalling induced significant elevation of Treg cell population in CIA and CAIA mice. We also demonstrated that inhibition of Notch signalling suppressed the progression of inflammatory arthritis through modulating the expansion and suppressive function of regulatory T (Treg) cells. Conclusion: Pharmacological and genetic inhibition of Notch1 signalling suppresses the progression of inflammatory arthritis through modulating the population and suppressive function of Treg cells in animal models of RA.

Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons

[Abstract] Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O2•−) and hydroxyl radical (•OH) and non-radical oxygen species such as hydrogen peroxide (H2O2), singlet oxygen (O2). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics (Ray et al., 2012). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer’s disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.

DRUG REPOSITIONING OF XHC FOR ALZHEIMER’S DISEASE: BACE1 PROMOTER REPRESSING ACTIVITY OF XHC

Background:Amyloid hypothesis postulated that exceed extracellular amyloid beta deposits are the fundamental cause of Alzheimer’s disease. Amyloid beta is produced by sequential proteolysis to amyloid beta precursor protein (APP) by beta-secretase (BACE1) and gamma-secretase. Another important phenomenon in AD patient is increased BACE1 expression. Methods:Our strategy is to find specific drugs reducing BACE1 expression rather than direct inhibition of BACE1. Using USA FDA approved drug library (Prestwick Chemical Library), we could discover putative therapeutic chemicals by cell based assay. Results:Among those candidates, XHC reduced the levels of BACE1 protein and mRNA in SH-SY5Y cells. A soluble APPb and C99 which are the products of BACE1 protease, were also decreased by treatment of XHC. We also confirmed that XHC could improve cognitive functions of 3XTg-AD mice. Decreased level of amyloid beta deposition and BACE1 expression also observed in XHC-treated AD mice. Conclusions: The fact that XHC is orally efficacious in AD animal models and is clinically safe to use make XHC an excellent candidate for advancement to clinical AD trials.

ROLE OF ADIPONECTIN IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE

Background: The thesis A causal nexus of mutually interacting elements exists; normal cognition requires their balance whereas imbalance creates AD. Interacting elements include APP, presenilins (PS), mitochondria, mitophagy, unfolded protein response (UPR), Wnt/ beta-catenin, cyclins, Notch, and calcium. In brief detail we describe, first, some effects of individual elements and, second, their mutual interactions.Methods:Literature review (citations will be provided). Results:A. Effects of individual components: Presenilins: Correct protein function requires protein-folding. PS1 plus excessive unfolded proteins, initiate the UPR; ATP is required for chaperones to assist protein-folding. Insufficient ATP from dysfunctional mitochondria causes improper protein-folding, leading to amyloid. PS1wt maintains gamma-secretase as inactive. Mutated/dysfunctional PS1(PSImut) activates gamma-secretase, which cleaves APP to generate A-beta. PS1wt also cleaves Notch1, thus affecting neuronal plasticity and cerebral vascular density. APP and Notch1 are competitive substrates for gamma-secretase so Notch1 activation reduces A-beta generation. High Notch1 levels in AD neurons may exist to improve the known, poor cerebral capillary vascularity in AD.Wnt 5a andWnt 7 signalling regulate adult neurogenesis in the SVZ, and also affect synapses;Wnt 3 affects differentiation of NSCs. Dysfunctional mitochondria in AD, major contributors to neuronal dysfunction, require elimination bymitophagy. Noteworthily, neuronal endocytosis initiates mitophagy before A-beta deposition. Mitophagy engulfs damaged mitochondria into autophagic vacuoles (AV); intermediate vacuoles develop, then fuse with lysosomes for digestion by cathepsins. AD brains showed 20-fold more AVs (they dispose impaired mitochondria, which stimulates biogenesis of normally functioning organelles). Calcium, whether high or low, stimulates autophagy. B. Interactions between elements of the causal nexus: Amyloid deposits in mitochondrial membranes create mitochondrial dysfunctions, decrease mitochondrial biogenesis, and impair neuronal function. A-beta causes low Wnt signaling, reducing neural b-catenin and neuronal viability. Interactions between PS1wt, cyclins and beta-catenin, disturb the neuronal cell cycle.PSImut producesmany impairments:WhereasPS1wt cleaves Notch1, PSImut inhibits Notch1, thereby reducing capillary density; PSImut enhances release of calcium from ER, causing impaired neuronal function; either high or low calcium stimulates mitophagy; PSImut inactivates elements required to promote UPR. Notch1&3 direct astrocyte formation from NHCs. Conclusions:Normal cognition requires balance within a nexus of interacting elements; imbalance would promote AD.