Jihoon Han

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.

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.

Fermented ginseng extract, BST204, disturbs adipogenesis of mesenchymal stem cells through inhibition of S6 kinase 1 signaling

Background The biological and pharmacological effects of BST204, a fermented ginseng extract, have been reported in various disease conditions. However, its molecular action in metabolic disease remains poorly understood. In this study, we identified the antiadipogenic activity of BST204 resulting from its inhibition of the S6 kinase 1 (S6K1) signaling pathway. Methods The inhibitory effects of BST204 on S6K1 signaling were investigated by immunoblot, nuclear fractionation, immunoprecipitation analyses. The antiadipogenic effect of BST204 was evaluated by measuring mRNA levels of adipogenic genes and by chromatin immunoprecipitation and quantitative real-time polymerase chain reaction analysis. Results Treatment with BST204 inhibited activation and nuclear translocation of S6K1, further decreasing the interaction between S6K1 and histone H2B in 10T1/2 mesenchymal stem cells. Subsequently, phosphorylation of H2B at serine 36 (H2BS36p) by S6K1 was reduced by BST204, inducing an increase in the mRNA expression of Wnt6, Wnt10a, and Wnt10b, which disturbed adipogenic differentiation and promoted myogenic and early osteogenic gene expression. Consistently, BST204 treatment during adipogenic commitment suppressed the expression of adipogenic marker genes and lipid drop formation. Conclusion Our results indicate that BST204 blocks adipogenesis of mesenchymal stem cells through the inhibition of S6K1-mediated histone phosphorylation. This study suggests the potential therapeutic strategy using BST204 to combat obesity and musculoskeletal diseases.

Reversine promotes browning of white adipocytes by suppressing miR-133a : KIM et al.

Brown adipocytes are characterized by a high number of uncoupling protein 1 (UCP1)-positive mitochondrial content and increased thermogenic capacity. As UCP1-enriched cells can consume lipids by generating heat, browning of white adipocytes is now highlighted as a promising approach for the prevention of obesity and obesity-associated metabolic diseases. Upon cold exposure or β-adrenergic stimuli, downregulation of microRNA-133 (miR-133) elevates the expression levels of PR domain containing 16 (Prdm16), which has been shown to be a brown adipose determination factor, in brown adipose tissue and subcutaneous white adipose tissues (WAT). Here, we show that treatment of reversine to white adipocytes induces browning via suppression of miR-133a. Reversine treatment promoted the expression of brown adipocyte marker genes, such as Prdm16 and UCP1, increasing the mitochondrial content, while decreasing the levels of miR-133a and white adipocyte marker genes. Ectopic expression of miR-133a mimic reversed the browning effects of the reversine treatment. Moreover, intraperitoneal administration of reversine in mice upregulated thermogenesis and resulted in resistance to high-fat diet-mediated weight gain as well as browning of subcutaneous and epididymal WAT. Taken together, we found a novel way to promote browning of white adipocytes through downregulation of miR-133a followed by activation of Prdm16, with a synthetic chemical, reversine.

S6K1 controls epigenetic plasticity for the expression of pancreatic α/β cell marker genes

The failure of insulin production by pancreatic β cells is a common hallmark of type 1 diabetes mellitus (T1DM). Because administration of exogenous insulin is associated with diabetes‐derived complications, endogenous α to β cell transition can be an attractive alternative. Although decreased β cell size and hypoinsulinaemia have been observed in S6K1‐deficient mice, the molecular mechanism underlying the involvement of S6K1 in the transcriptional regulation of insulin remains elusive. Here, we show that the hypoinsulinaemic phenotype of S6K1‐deficient mice stems from the dysregulated transcription of a set of genes required for insulin and glucagon production. First, we observed that increased expression of α cell marker genes and decreased expression of β cell marker genes in pancreas tissues from S6K1‐deficient mice. Furthermore, S6K1 was highly activated in murine β cell line, βTC6, compared to murine α cell line αTC1. In both α and β cells, active S6K1 promoted the transcription of β cell marker genes, including insulin, whereas S6K1 inhibition increased the transcription of α cell marker genes. Moreover, S6K1 mediated pancreatic gene regulation by modifying two histone marks (activating H3K4me3 and repressing H3K27me3) on gene promoters. These results suggest that S6K1 drives the α to β transition through the epigenetic regulation of cell‐specific genes, including insulin and glucagon. This novel role of S6K1 in islet cells provides basic clues to establish therapeutic strategies against T1DM.

Epigenetic role of nuclear S6K1 in early adipogenesis

S6K1 is a key regulator of cell growth, cell size, and metabolism. Although the role of cytosolic S6K1 in cellular processes is well established, the function of S6K1 in the nucleus remains poorly understood. Our recent study has revealed that S6K1 is translocated into the nucleus upon adipogenic stimulus where it directly binds to and phosphorylates H2B at serine 36. Such phosphorylation promotes EZH2 recruitment and subsequent histone H3K27 trimethylation on the promoter of its target genes including Wnt6, Wnt10a, and Wnt10b, leading to repression of their expression. S6K1-mediated suppression of Wnt genes facilitates adipogenic differentiation through the expression of adipogenic transcription factors PPARγ and Cebpa. White adipose tissues from S6K1-deficient mice consistently exhibit marked reduction in H2BS36 phosphorylation (H2BS36p) and H3K27 trimethylation (H3K27me3), leading to enhanced expression of Wnt genes. In addition, expression levels of H2BS36p and H3K27me3 are highly elevated in white adipose tissues from mice fed on high-fat diet or from obese humans. These findings describe a novel role of S6K1 as a transcriptional regulator controlling an epigenetic network initiated by phosphorylation of H2B and trimethylation of H3, thus shutting off Wnt gene expression in early adipogenesis. [BMB Reports 2016; 49(8): 401-402]

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.