Márcia M. Aranha

miR-34a Regulates Mouse Neural Stem Cell Differentiation

Background MicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation. Methodology/Principal Findings miR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism. Conclusions Our results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.

Bile acid levels are increased in the liver of patients with steatohepatitis

Background/aims The pathogenesis of steatohepatitis remains largely unknown; however, bile acids may play a role as potential mediators of liver damage. The aim of this study was to characterize bile acid profiles in liver tissue of patients with steatohepatitis. Methods Bile acid composition was determined by gas–liquid chromatography in liver tissue from patients with nonalcoholic steatohepatitis (NASH; n=15), patients with alcoholic steatohepatitis (ASH; n=14), and controls (n=8). Liver biopsies were graded for steatosis, inflammation, and fibrosis. Results Bile acids were moderately increased in liver tissue of steatohepatitis patients compared with controls (P<0.05). Deoxycholic, chenodeoxycholic, and cholic acids were elevated by 92, 64, and 43%, respectively, in patients with steatohepatitis (P<0.05). Cholic acid was the prevailing bile acid in NASH patients and in controls. More hydrophobic bile acid species were elevated in ASH patients compared with controls (P<0.05). Significant correlations were found in NASH patients between hepatic chenodeoxycholic acid and fibrosis, and between cholic acid and trihydroxy/dihydroxy bile acids and inflammation (P<0.05). In patients with ASH, cholic acid and trihydroxy/dihydroxy bile acids were correlated with steatosis (P<0.01). Conclusion This study shows a distinct pattern of bile acids in the liver of patients with steatohepatitis. Further, the association between bile acids and histological liver injury suggests an association of specific bile acids and disease progression, possibly through bile acid-induced liver injury.

Apoptosis-associated microRNAs are modulated in mouse, rat and human neural differentiation

BackgroundMicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However, evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently, we have shown that apoptosis-associated factors, such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development, we performed global miRNA expression profiling experiments in NS cells. Next, we characterized the expression of proapoptotic miRNAs, including miR-16, let-7a and miR-34a in distinct models of neural differentiation, including mouse embryonic stem cells, PC12 and NT2N cells. In addition, the expression of antiapoptotic miR-19a and 20a was also evaluated.ResultsThe expression of miR-16, let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast, expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly, differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells.ConclusionsIn conclusion, the identification of miR-16, let-7a and miR-34a, whose expression patterns are conserved in mouse, rat and human neural differentiation, implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs.

Safety, tolerability, and cerebrospinal fluid penetration of ursodeoxycholic acid in patients with amyotrophic lateral sclerosis

Objective:Amyotrophic lateral sclerosis is a progressive degenerative disease, which typically leads to death in 3 to 5 years. Neuronal cell death offers a potential target for therapeutic intervention. Ursodeoxycholic acid is a cytoprotective, endogenous bile acid that has been shown to be neuroprotective in experimental Huntington and Alzheimer diseases, retinal degeneration, and ischemic and hemorrhagic stroke. The objective of this research was to study the safety and the tolerability of ursodeoxycholic acid in amyotrophic lateral sclerosis and document effective and dose-dependent cerebrospinal fluid penetration. Methods:Eighteen patients were randomly assigned to receive ursodeoxycholic acid at doses of 15, 30, and 50 mg/kg of body weight per day. Serum and cerebrospinal fluid were obtained for analysis after 4 weeks of treatment. Treatment-emergent clinical and laboratory events were monitored weekly. Results:Our data indicated that ursodeoxycholic acid is well tolerated by all subjects at all doses. We also showed that ursodeoxycholic acid is well absorbed after oral administration and crosses the blood-brain barrier in a dose-dependent manner. Conclusions:These results show excellent safety and tolerability of ursodeoxycholic acid. The drug penetrates the cerebrospinal fluid in a dose-dependent manner. A large, placebo-controlled clinical trial is needed to assess the efficacy of ursodeoxycholic acid in treating amyotrophic lateral sclerosis.

p53 interaction with JMJD3 results in its nuclear distribution during mouse neural stem cell differentiation.

Conserved elements of apoptosis are also integral components of cellular differentiation. In this regard, p53 is involved in neurogenesis, being required for neurite outgrowth in primary neurons and for axonal regeneration in mice. Interestingly, demethylases regulate p53 activity and its interaction with co-activators by acting on non-histone proteins. In addition, the histone H3 lysine 27-specific demethylase JMJD3 induces ARF expression, thereby stabilizing p53 in mouse embryonic fibroblasts. We hypothesized that p53 interacts with key regulators of neurogenesis to redirect stem cells to differentiation, as an alternative to cell death. Specifically, we investigated the potential cross-talk between p53 and JMJD3 during mouse neural stem cell (NSC) differentiation. Our results demonstrated that JMJD3 mRNA and protein levels were increased early in mouse NSC differentiation, when JMJD3 activity was readily detected. Importantly, modulation of JMJD3 in NSCs resulted in changes of total p53 protein, coincident with increased ARF mRNA and protein expression. ChIP analysis revealed that JMJD3 was present at the promoter and exon 1 regions of ARF during neural differentiation, although without changes in H3K27me3. Immunoprecipitation assays demonstrated a direct interaction between p53 and JMJD3, independent of the C-terminal region of JMJD3, and modulation of p53 methylation by JMJD3-demethylase activity. Finally, transfection of mutant JMJD3 showed that the demethylase activity of JMJD3 was crucial in regulating p53 cellular distribution and function. In conclusion, JMJD3 induces p53 stabilization in mouse NSCs through ARF-dependent mechanisms, directly interacts with p53 and, importantly, causes nuclear accumulation of p53. This suggests that JMJD3 and p53 act in a common pathway during neurogenesis.

NF‐κB and apoptosis in colorectal tumourigenesis

Background  Nuclear factor‐κB (NF‐κB) may play an important role in colorectal tumourigenesis, controlling cell cycle and apoptosis gene expression. In addition, imbalances between cell proliferation and cell death are thought to underlie neoplastic development. The aims of this study were to investigate apoptosis and expression of several apoptosis‐related proteins, and to determine correlations with colorectal tumour progression.

Caspases and p53 Modulate FOXO3A/Id1 Signaling During Mouse Neural Stem Cell Differentiation

Neural stem cells (NSCs) differentiate into neurons and glia, and a large percentage undergoes apoptosis. The engagement and activity of apoptotic pathways may favor either cell death or differentiation. In addition, Akt represses differentiation by up‐regulating the inhibitor of differentiation 1 (Id1), through phosphorylation of its repressor FOXO3A. The aim of this study was to investigate the potential cross‐talk between apoptosis and proliferation during mouse NSC differentiation. We determined the time of neurogenesis and gliogenesis using neuronal β‐III tubulin and astroglial GFAP to confirm that both processes occurred at ∼3 and 8 days, respectively. p‐Akt, p‐FOXO3A, and Id1 were significantly reduced throughout differentiation. Caspase‐3 processing, p53 phosphorylation, and p53 transcriptional activation increased at 3 days of differentiation, with no evidence of apoptosis. Importantly, in cells exposed to the pancaspase inhibitor z‐VAD.fmk, p‐FOXO3A and Id1 were no longer down‐regulated, p53 phosphorylation and transcriptional activation were reduced, while neurogenesis and gliogenesis were significantly delayed. The effect of siRNA‐mediated silencing of p53 on FOXO3A/Id1 was similar to that of z‐VAD.fmk only at 3 days of differentiation. Interestingly, caspase inhibition further increased the effect of p53 knockdown during neurogenesis. In conclusion, apoptosis‐associated factors such as caspases and p53 temporally modulate FOXO3A/Id1 signaling and differentiation of mouse NSCs. J. Cell. Biochem. 107: 748–758, 2009.

Driving Apoptosis-relevant Proteins Toward Neural Differentiation

Emerging evidence suggests that apoptosis regulators and executioners may control cell fate, without involving cell death per se. Indeed, several conserved elements of apoptosis are integral components of terminal differentiation, which must be restrictively activated to assure differentiation efficiency, and carefully regulated to avoid cell loss. A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation, as an alternative to cell death will surely make stem cells more suitable for neuro-replacement therapies. In this review, we summarize recent studies on the mechanisms underlying the non-apoptotic function of p53, caspases, and Bcl-2 family members during neural differentiation. In addition, we discuss how apoptosis-regulatory proteins control the decision between differentiation, self-renewal, and cell death in neural stem cells, and how activity is restrained to prevent cell loss.

Colorectal cancer: Can nutrients modulate NF-κB and apoptosis?

BACKGROUND & AIMS NF-kappaB may promote carcinogenesis by altering cell cycle, inflammatory responses and apoptosis-related gene expression, though cell mechanisms relating diet and colorectal cancer (CRC) remain unveiled in humans. This study in patients with CRC aimed to explore potential interactions between the dietary pattern, nutrient intake, expression of NF-kappaB, apoptosis and tumour histological aggressiveness. METHODS Usual diet was assessed by diet history; nutrient composition was determined by DIETPLAN software. Histologically classified patient tissue samples (adenoma, adenocarcinoma and normal surrounding mucosa) were obtained via biopsies during colonoscopy (n=16) or surgery (n=8). NF-kappaB expression was determined by immunohistochemistry and apoptosis by TUNEL assay. RESULTS NF-kappaB expression and apoptosis were higher in tumours (p<0.01), greater along with histological aggressiveness (p<0.01). Highest intake terciles of animal protein, refined carbohydrates, saturated fat, n-6 fatty acids and alcohol were associated with higher NF-kappaB, apoptosis and histological aggressiveness (p<0.01); the opposite tissue characteristics were associated with highest intake terciles of n-3 fatty acids, fibre, vitamin E, flavonoids, isoflavones, beta-carotene and selenium (p<0.002). Additionally, higher n-6:n-3 fatty acids ratio (median 26:1) was associated with higher NF-kappaB (p<0.006) and apoptosis (p<0.01), and more aggressive histology (p<0.01). Conversely, lower n-6:n-3 fatty acids ratio (median 6:1) was associated with lower NF-kappaB (p<0.002) and apoptosis (p<0.002), and less aggressive histology (p<0.002). CONCLUSIONS NF-kappaB expression and apoptosis increased from adenoma to poorly differentiated adenocarcinoma. This degenerative transition, recognized as key in carcinogenesis, appear to have been influenced by a diet promoting a pro-inflammatory milieu that can trigger NF-kappaB.