Downregulation of CREB expression in Alzheimer's brain and in Aβ-treated rat hippocampal neurons

<p>Abstract</p> <p>Background</p> <p>Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and Aβ. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post-mortem brain and in cultured rat hippocampal neurons exposed to Aβ aggregates.</p> <p>Results</p> <p>Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS-extracted Aβ and CREB protein levels in Alzheimer's post-mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to Aβ fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in Aβ-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against Aβ-induced neuronal apoptosis.</p> <p>Conclusions</p> <p>Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression.</p

Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells

Human islets isolated for transplantation are exposed to multiple stresses including oxidative stress and hypoxia resulting in significant loss of functional β cell mass. In this study we examined the modulation of apoptosis pathway genes in islets exposed to hydrogen peroxide, peroxynitrite, hypoxia, and cytokines. We observed parallel induction of pro- and antiapoptotic pathways and identified several novel genes including BFAR, CARD8, BNIP3, and CIDE-A. As BNIP3 is an inducer of autophagy, we examined this pathway in MIN6 cells, a mouse beta cell line and in human islets. Culture of MIN6 cells under low serum conditions increased the levels of several proteins in autophagy pathway, including ATG4, Beclin 1, LAMP-2, and UVRAG. Amino acid deprivation led to induction of autophagy in human islets. Preconditioning of islets with inducers of autophagy protected them from hypoxia-induced apoptosis. However, induction of autophagy during hypoxia exacerbated apoptotic cell death. ER stress led to induction of autophagy and apoptosis in β cells. Overexpression of MnSOD, an enzyme that scavenges free radicals, resulted in protection of MIN6 cells from cytokine-induced apoptosis. Ceramide, a mediator of cytokine-induced injury, reduced the active phosphorylated form of Akt and downregulated the promoter activity of the antiapoptotic gene bcl-2. Furthermore, cytokine-stimulated JNK pathway downregulated the bcl-2 promoter activity which was reversed by preincubation with SP600125, a JNK inhibitor. Our findings suggest that β cell apoptosis by multiple stresses in islets isolated for transplantation is the result of orchestrated gene expression in apoptosis pathway

Synergistic induction of heme oxygenase-1 by the components of the antioxidant supplement Protandim. Free Radic.

Protandim is an antioxidant supplement that consists of five ingredients, namely, ashwagandha, bacopa extract, green tea extract, silymarin, and curcumin, each with known therapeutic properties. Protandim was formulated with the objective of combining multiple phytochemicals at low nontoxic doses to gain synergy among them. A recent clinical study demonstrated the in vivo antioxidant effects of Protandim (S.K. Nelson et al., 2006, Free Radic. Biol. Med. 40, 341-347). The objective of the present study was to determine if the components of Protandim induce heme oxygenase-1 (HO-1) in a synergistic manner in cultured MIN6 cells, a mouse β-cell line, and in SK-N-MC cells, a human neuroblastoma cell line. When the components of Protandim were tested alone at low doses, curcumin showed minimal induction, whereas the others were unable to induce the HO-1 promoter, assayed by transient transfection. All components together, however, produced a strongly synergistic induction of around three-to ninefold in a dose-dependent manner, greatly exceeding the sum of the parts. Similar findings were obtained for the expression of HO-1 at the mRNA and protein levels. Protandim-mediated HO-1 induction involved the presence of ARE sites in the HO-1 promoter and nuclear translocalization of the transcription factor Nrf2, which binds to ARE sites. The involvement of multiple signaling pathways, including PI3-kinase/Akt, p38MAPK, and PKCδ, in HO-1 induction seems to be the probable mechanism of synergy between the components of Protandim. There were significant increases in the levels of total glutathione in Protandimtreated cells. These findings suggest that the use of a combination of phytochemicals may be an efficient method for the induction of antioxidant enzymes

Synergistic induction of heme oxygenase-1 by the components of the antioxidant supplement Protandim. Free Radic.

Protandim is an antioxidant supplement that consists of five ingredients, namely, ashwagandha, bacopa extract, green tea extract, silymarin, and curcumin, each with known therapeutic properties. Protandim was formulated with the objective of combining multiple phytochemicals at low nontoxic doses to gain synergy among them. A recent clinical study demonstrated the in vivo antioxidant effects of Protandim (S.K. Nelson et al., 2006, Free Radic. Biol. Med. 40, 341-347). The objective of the present study was to determine if the components of Protandim induce heme oxygenase-1 (HO-1) in a synergistic manner in cultured MIN6 cells, a mouse β-cell line, and in SK-N-MC cells, a human neuroblastoma cell line. When the components of Protandim were tested alone at low doses, curcumin showed minimal induction, whereas the others were unable to induce the HO-1 promoter, assayed by transient transfection. All components together, however, produced a strongly synergistic induction of around three-to ninefold in a dose-dependent manner, greatly exceeding the sum of the parts. Similar findings were obtained for the expression of HO-1 at the mRNA and protein levels. Protandim-mediated HO-1 induction involved the presence of ARE sites in the HO-1 promoter and nuclear translocalization of the transcription factor Nrf2, which binds to ARE sites. The involvement of multiple signaling pathways, including PI3-kinase/Akt, p38MAPK, and PKCδ, in HO-1 induction seems to be the probable mechanism of synergy between the components of Protandim. There were significant increases in the levels of total glutathione in Protandimtreated cells. These findings suggest that the use of a combination of phytochemicals may be an efficient method for the induction of antioxidant enzymes

Dramatic Co-Activation of WWOX/WOX1 with CREB and NF-κB in Delayed Loss of Small Dorsal Root Ganglion Neurons upon Sciatic Nerve Transection in Rats

BACKGROUND:Tumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats. METHODOLOGY/PRINCIPAL FINDINGS:Sciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-kappaB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-kappaB (40-65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-kappaB. WOX1 directly activated NF-kappaB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons. CONCLUSIONS/SIGNIFICANCE:Rapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-kappaB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-kappaB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury

A Promising Strategy to Treat Neurodegenerative Diseases by SIRT3 Activation

SIRT3, the primary mitochondrial deacetylase, regulates the functions of mitochondrial proteins including metabolic enzymes and respiratory chain components. Although SIRT3’s functions in peripheral tissues are well established, the significance of its downregulation in neurodegenerative diseases is beginning to emerge. SIRT3 plays a key role in brain energy metabolism and provides substrate flexibility to neurons. It also facilitates metabolic coupling between fuel substrate-producing tissues and fuel-consuming tissues. SIRT3 mediates the health benefits of lifestyle-based modifications such as calorie restriction and exercise. SIRT3 deficiency is associated with metabolic syndrome (MetS), a precondition for diseases including obesity, diabetes, and cardiovascular disease. The pure form of Alzheimer’s disease (AD) is rare, and it has been reported to coexist with these diseases in aging populations. SIRT3 downregulation leads to mitochondrial dysfunction, neuroinflammation, and inflammation, potentially triggering factors of AD pathogenesis. Recent studies have also suggested that SIRT3 may act through multiple pathways to reduce plaque formation in the AD brain. In this review, we give an overview of SIRT3’s roles in brain physiology and pathology and discuss several activators of SIRT3 that can be considered potential therapeutic agents for the treatment of dementia