Abstract: Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's 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 Alzheimer's 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.

Abstract: Our studies have revealed an unrecognized mechanism for regulating the activity of p53 during the cell cycle. We show, using multiple approaches, that p53 is degraded as a consequence of its phosphorylation by Aurora B. This degradation ultimately prevents p53 from performing its role as a guardian of the genome. Taken together with our earlier study on the effects of the Aurora B kinase inhibitor AZD1152 on breast cancer cells and preclinical evaluations of this drug for treating diverse human tumors, we conclude that Aurora B inhibitors may prove to be effective in treating cancer cells expressing functional p53 molecules. Also, we previously showed that specific inhibition of Aurora B expression is sufficient to inhibit tumor growth and induce the regression of tumors. Significantly, we here show that a specific inhibitor (AZD1152) of Aurora B can cause p53 elevation, thereby increasing p53 target gene expression in a cancer xenograft model. As a result, inhibitor of Aurora B can reduce cell survival through increasing the p53 up-regulated mediator of apoptosis p53 up-regulated modulator of apoptosis (PUMA) and cause cell cycle inhibition through inducing CDK inhibitor p21. These data are consistent with biochemical studies in cell lines. Our present study defines the molecular mechanisms involved in inhibition of tumor growth by an Aurora B inhibitor. General suppression of transcription is observed during mitosis ( 4 ); therefore, Aurora B-mediated p53 transcriptional suppression will not play a role during mitosis. The work by Cross et al. ( 2 ) shows that p53 is involved in facilitating chromosome segregation to ensure the maintenance of diploid cells. Aurora B may coordinate with p53 to mediate the spindle checkpoint ( 2 ) and aid progression through mitosis. Given that Aurora B deregulation also results in polyploidy, the interplay between p53 and Aurora B is conceivably important for spindle checkpoint. The functional significance of Aurora B–p53 interaction during different stages of mitosis remains to be investigated, but p53 is involved in spindle checkpoint ( 2 ); our data serve to confirm its presence in the spindle checkpoint machinery. It is important to point out that the binding between Aurora B and p53 decreases after the end of mitosis because of the down-regulation of Aurora B by E3 ubiquitin ligase anaphase promoting complex. After mitosis, Aurora B is recovering from degradation and again binding p53, and thus, it potentially prevents p53 from arresting the cell cycle at G1 by maintaining a negative impact on p53. The enhanced degradation of phosphorylated p53 leads to the finding that its transcriptional activity on cell cycle control gene was diminished (such as p21 CDK inhibitor) when Aurora B was overexpressed. Moreover, we were able to show that p53-mediated gene transcription was enhanced when Aurora B expression was inhibited. These data provide a rationale for the role of Aurora B in interphase, which is to antagonize the inhibition of cell cycle progression by p53 and allow entry of cells into mitosis. To answer this question, we used bimolecular fluorescence complementation, which allows direct observation of the interaction between two proteins in living cells, and showed that Aurora B and p53 interact during interphase and mitosis ( Fig. P1 ). Immunofluorescence studies showed that, during mitosis, p53 associates with Aurora B located at centromeres at prometaphase or the middle zone of the cleavage furrow at the anaphase–telophase border. We were also able to show that Survivin, a protein component of CPC localized at the centromeres to activate Aurora B ( 3 ), colocalized with Aurora B and p53 to the DNA of prometaphase cells. We also showed that Aurora B and p53 could be coimmunoprecipitated with specific antibodies. Using the technique, we were also able to show the Aurora B–p53 association in every phase of the cell cycle except late M phase, when Aurora B is known to be degraded. The interaction in interphase is quite surprising, because Aurora B has been shown to function exclusively during mitosis. We then showed that Aurora B phosphorylates p53 at three predicted Aurora B sites (S183, T211, and S215). We also showed that these phosphorylations lead to enhanced degradation of p53 through ubiquitination, which is mediated by the murine double minute 2 (MDM2) regulatory protein, an E3 ubiquitin ligase of p53. Moreover, AZD1152-hydroxyquinazoline pyrazole anilide (HQPA), which specifically inhibits the kinase activity of Aurora B, was able to block p53 ubiquitination in a dose-dependent manner. These data provide insights into the contribution of Aurora B-mediated p53 phosphorylation in regulating p53 stability. The regulation of mitosis ensures the equal segregation of chromosomes to daughter cells, and Aurora B plays a critical role in this process as a component of the chromosomal passenger protein complex (CPC). CPC is located on the chromosome arms during prophase and at the centromeres during prometaphase and metaphase ( 1 ). Aurora B subsequently localizes to the midbody during cytokinesis and participates in ensuring the correct attachment of chromosomes to spindle microtubules (spindle checkpoint) and equal distribution of chromosomes. The effects of Aurora B are exerted by its phosphorylation on specific proteins of the mitotic apparatus. The p53 protein is also involved in facilitating chromosome segregation to ensure the maintenance of diploid cells because cells deficient in p53 expression become tetraploid ( 2 ). Thus, it raises the question of whether Aurora B and p53 are functionally related. Aurora B, a protein kinase, plays a key role in mitosis by maintaining correct chromosome segregation and progression of cells through mitosis. Cancer cells frequently express Aurora B at unusually high levels, leading to dysregulated mitosis and therefore, causing unequal chromosome segregation, which may confer a growth advantage. The tumor suppressor protein p53 guards the genome by delaying or arresting the cell cycle at specific checkpoints (G1/S) when DNA is damaged and prevents damaged cells from entering mitosis (G2/M checkpoint). Despite intensive studies on p53 for more than three decades, the exact mechanism by which it regulates mitotic checkpoint is unknown. Whether Aurora B and p53 are coordinately regulated during the cell cycle remains to be determined, and there is no report to our knowledge suggesting that Aurora B functions in processes other than mitosis. By studying cells synchronized to divide at the same time, we show here that Aurora B and p53 interact during all stages of the cell cycle except during late M phase, when Aurora B is degraded. Moreover, we show that Aurora B is a negative regulator of p53 and that, when overexpressed, it affects the ability of p53 to mitigate the detrimental consequences of DNA damage and control the mitotic checkpoint.

Abstract: Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90 N (TRAP1 N ) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis -bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.

Abstract: Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo–cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep -Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep- Tactin but no longer requires Hsp70/40. Consistent with a Strep -Tactin–dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.

Abstract: This study investigates how native speakers and L2 learners of English (L2 learners being native speakers of Korean) produce voiceless stops in English in the following phonological contexts: word-initial vs. -medial, stressed vs. unstressed, and when preceded by /s/. The study also examines the correlation between proficiency of L2 learners of English and the degree of aspiration (represented by VOT) in English voiceless stops. The speech of Korean L2 learners of English rated and categorized into 5 English proficiency levels in Genie Speech Corpus will be used. We will measure VOT of voiceless stops in the aforementioned conditions produced by 5 English speakers and each 5 Korean speakers per level. We expect that highly rated L2 speakers of English will produce longer VOT in stressed syllables than in unstressed syllables, as native speakers of English do. However, as the proficiency level gets lower, L2 speakers will produce it to a lesser degree than native speakers do, presenting a wide variety of VOT length.