Squamocin modulates histone H3 phosphorylation levels and induces G1 phase arrest and apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. Increased phosphorylation levels of histone H3 as a result of aurora B and pMSK1 overexpression were observed in various tumors. We selected <it>aurora B </it>and <it>MSK1 </it>as representatives for testing various compounds and drugs, and found that squamocin, a bis-tetrahydrofuran annonaceous acetogenin, exerted a potent effect on histone H3 phosphorylation.</p> <p>Methods</p> <p>GBM8401, Huh-7, and SW620 cells were incubated with 15, 30, and 60 μM squamocin for 24 h. The expressions of mRNA and proteins were analyzed by qRT-PCR and Western blotting, respectively. The cell viability was determined by an MTT assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry.</p> <p>Results</p> <p>Our results showed that squamocin inhibited the proliferation of GBM8401, Huh-7, and SW620 cells, arrested the cell cycle at the G₁phase, and activated both intrinsic and extrinsic pathways to apoptosis. In addition, we demonstrated that squamocin had the ability to modulate the phosphorylation levels of H3S10 (H3S10p) and H3S28 (H3S28p) in association with the downregulation of aurora B and pMSK1 expressions.</p> <p>Conclusions</p> <p>This study is the first to show that squamocin affects epigenetic alterations by modulating histone H3 phosphorylation at S10 and S28, providing a novel view of the antitumor mechanism of squamocin.</p

Severity dependent distribution of impairments in PSP and CBS: Interactive visualizations

BACKGROUND: Progressive supranuclear palsy (PSP) -Richardson's Syndrome and Corticobasal Syndrome (CBS) are the two classic clinical syndromes associated with underlying four repeat (4R) tau pathology. The PSP Rating Scale is a commonly used assessment in PSP clinical trials; there is an increasing interest in designing combined 4R tauopathy clinical trials involving both CBS and PSP. OBJECTIVES: To determine contributions of each domain of the PSP Rating Scale to overall severity and characterize the probable sequence of clinical progression of PSP as compared to CBS. METHODS: Multicenter clinical trial and natural history study data were analyzed from 545 patients with PSP and 49 with CBS. Proportional odds models were applied to model normalized cross-sectional PSP Rating Scale, estimating the probability that a patient would experience impairment in each domain using the PSP Rating Scale total score as the index of overall disease severity. RESULTS: The earliest symptom domain to demonstrate impairment in PSP patients was most likely to be Ocular Motor, followed jointly by Gait/Midline and Daily Activities, then Limb Motor and Mentation, and finally Bulbar. For CBS, Limb Motor manifested first and ocular showed less probability of impairment throughout the disease spectrum. An online tool to visualize predicted disease progression was developed to predict relative disability on each subscale per overall disease severity. CONCLUSION: The PSP Rating Scale captures disease severity in both PSP and CBS. Modelling how domains change in relation to one other at varying disease severities may facilitate detection of therapeutic effects in future clinical trials

A miRNA Signature of Prion Induced Neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified

Molecular Basis for Vulnerability to Mitochondrial and Oxidative Stress in a Neuroendocrine CRI-G1 Cell Line

Many age-associated disorders (including diabetes, cancer, and neurodegenerative diseases) are linked to mitochondrial dysfunction, which leads to impaired cellular bioenergetics and increased oxidative stress. However, it is not known what genetic and molecular pathways underlie differential vulnerability to mitochondrial dysfunction observed among different cell types.Starting with an insulinoma cell line as a model for a neuronal/endocrine cell type, we isolated a novel subclonal line (named CRI-G1-RS) that was more susceptible to cell death induced by mitochondrial respiratory chain inhibitors than the parental CRI-G1 line (renamed CRI-G1-RR for clarity). Compared to parental RR cells, RS cells were also more vulnerable to direct oxidative stress, but equally vulnerable to mitochondrial uncoupling and less vulnerable to protein kinase inhibition-induced apoptosis. Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery. Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1) inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2) decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3) increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4) increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5) reconstitution of glucose-stimulated insulin secretion.The molecular profile presented here will enable identification of individual genes or gene clusters that shape vulnerability to mitochondrial dysfunction and thus represent potential therapeutic targets for diabetes and neurodegenerative diseases. In addition, the newly identified CRI-G1-RS cell line represents a new experimental model for investigating how endogenous antioxidants affect glucose sensing and insulin release by pancreatic β cells

Copy number elevation of 22q11.2 genes arrests the developmental maturation of working memory capacity and adult hippocampal neurogenesis

Working memory capacity, a critical component of executive function, expands developmentally from childhood through adulthood. Anomalies in this developmental process are seen in individuals with autism spectrum disorder (ASD), schizophrenia and intellectual disabilities (ID), implicating this atypical process in the trajectory of developmental neuropsychiatric disorders. However, the cellular and neuronal substrates underlying this process are not understood. Duplication and triplication of copy number variants of 22q11.2 are consistently and robustly associated with cognitive deficits of ASD and ID in humans, and overexpression of small 22q11.2 segments recapitulates dimensional aspects of developmental neuropsychiatric disorders in mice. We capitalized on these two lines of evidence to delve into the cellular substrates for this atypical development of working memory. Using a region- and cell-type-selective gene expression approach, we demonstrated that copy number elevations of catechol-O-methyl-transferase (COMT) or Tbx1, two genes encoded in the two small 22q11.2 segments, in adult neural stem/progenitor cells in the hippocampus prevents the developmental maturation of working memory capacity in mice. Moreover, copy number elevations of COMT or Tbx1 reduced the proliferation of adult neural stem/progenitor cells in a cell-autonomous manner in vitro and migration of their progenies in the hippocampus granular layer in vivo. Our data provide evidence for the novel hypothesis that copy number elevations of these 22q11.2 genes alter the developmental trajectory of working memory capacity via suboptimal adult neurogenesis in the hippocampus.Peer reviewe

Gene Expression Profiles in Parkinson Disease Prefrontal Cortex Implicate FOXO1 and Genes under Its Transcriptional Regulation

Parkinson disease (PD) is a complex neurodegenerative disorder with largely unknown genetic mechanisms. While the degeneration of dopaminergic neurons in PD mainly takes place in the substantia nigra pars compacta (SN) region, other brain areas, including the prefrontal cortex, develop Lewy bodies, the neuropathological hallmark of PD. We generated and analyzed expression data from the prefrontal cortex Brodmann Area 9 (BA9) of 27 PD and 26 control samples using the 44K One-Color Agilent 60-mer Whole Human Genome Microarray. All samples were male, without significant Alzheimer disease pathology and with extensive pathological annotation available. 507 of the 39,122 analyzed expression probes were different between PD and control samples at false discovery rate (FDR) of 5%. One of the genes with significantly increased expression in PD was the forkhead box O1 (FOXO1) transcription factor. Notably, genes carrying the FoxO1 binding site were significantly enriched in the FDR–significant group of genes (177 genes covered by 189 probes), suggesting a role for FoxO1 upstream of the observed expression changes. Single-nucleotide polymorphisms (SNPs) selected from a recent meta-analysis of PD genome-wide association studies (GWAS) were successfully genotyped in 50 out of the 53 microarray brains, allowing a targeted expression–SNP (eSNP) analysis for 52 SNPs associated with PD affection at genome-wide significance and the 189 probes from FoxO1 regulated genes. A significant association was observed between a SNP in the cyclin G associated kinase (GAK) gene and a probe in the spermine oxidase (SMOX) gene. Further examination of the FOXO1 region in a meta-analysis of six available GWAS showed two SNPs significantly associated with age at onset of PD. These results implicate FOXO1 as a PD–relevant gene and warrant further functional analyses of its transcriptional regulatory mechanisms

PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation

Efficient RT-QuIC seeding activity for \u3b1-synuclein in olfactory mucosa samples of patients with Parkinson's disease and multiple system atrophy

Background: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal \u3b1-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either \u3b1-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of \u3b1-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM). Methods: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant \u3b1-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce \u3b1-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP. Results: Our results showed that a significant percentage of MSA and PD samples induced \u3b1-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination. Conclusions: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for \u3b1-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages

Inactivation of CDK/pRb Pathway Normalizes Survival Pattern of Lymphoblasts Expressing the FTLD-Progranulin Mutation c.709-1G>A

8 figuras, 2 tablasBackground Mutations in the progranulin (PGRN) gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP), although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK) CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation. Methodology/Principal Findings We performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal. Conclusion/Significance The use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDPThis work has been supported by grants from Ministry of Education and Science (SAF2007-61701, SAF2010-15700, SAF2011-28603), Fundación Eugenio Rodríguez Pascual, and Basque Government (Saiotek program 2008–2009). NE holds a fellowship of the JAE predoctoral program of the CSICPeer reviewe

Re-cycling paradigms: cell cycle regulation in adult hippocampal neurogenesis and implications for depression

Since adult neurogenesis became a widely accepted phenomenon, much effort has been put in trying to understand the mechanisms involved in its regulation. In addition, the pathophysiology of several neuropsychiatric disorders, such as depression, has been associated with imbalances in adult hippocampal neurogenesis. These imbalances may ultimately reflect alterations at the cell cycle level, as a common mechanism through which intrinsic and extrinsic stimuli interact with the neurogenic niche properties. Thus, the comprehension of these regulatory mechanisms has become of major importance to disclose novel therapeutic targets. In this review, we first present a comprehensive view on the cell cycle components and mechanisms that were identified in the context of the homeostatic adult hippocampal neurogenic niche. Then, we focus on recent work regarding the cell cycle changes and signaling pathways that are responsible for the neurogenesis imbalances observed in neuropathological conditions, with a particular emphasis on depression