Ming-Chang Chiang

The neuroprotective role of metformin in advanced glycation end product treated human neural stem cells is AMPK-dependent.

Diabetic neuronal damage results from hyperglycemia followed by increased formation of advanced glycosylation end products (AGEs), which leads to neurodegeneration, although the molecular mechanisms are still not well understood. Metformin, one of the most widely used anti-diabetic drugs, exerts its effects in part by activation of AMP-activated protein kinase (AMPK). AMPK is a critical evolutionarily conserved enzyme expressed in the liver, skeletal muscle and brain, and promotes cellular energy homeostasis and biogenesis by regulating several metabolic processes. While the mechanisms of AMPK as a metabolic regulator are well established, the neuronal role for AMPK is still unknown. In the present study, human neural stem cells (hNSCs) exposed to AGEs had significantly reduced cell viability, which correlated with decreased AMPK and mitochondria associated gene/protein (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3 and 9 activities. Metformin prevented AGEs induced cytochrome c release from mitochondria into cytosol in the hNSCs. Co-treatment with metformin significantly abrogated the AGE-mediated effects in hNSCs. Metformin also significantly rescued hNSCs from AGE-mediated mitochondrial deficiency (lower ATP, D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Furthermore, co-treatment of hNSCs with metformin significantly blocked AGE-mediated reductions in the expression levels of several neuroprotective genes (PPARγ, Bcl-2 and CREB). These findings extend our understanding of the molecular mechanisms of both AGE-induced neuronal toxicity, and AMPK-dependent neuroprotection by metformin. This study further suggests that AMPK may be a potential therapeutic target for treating diabetic neurodegeneration.

PPARγ regulates the mitochondrial dysfunction in human neural stem cells with tumor necrosis factor alpha

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated transcription factors, and its ligands are known to control many physiological and pathological conditions. The hypothesis of our study was that the PPARγ agonist (rosiglitazone) could mediate tumor necrosis factor alpha (TNFα) related to the regulation of human neural stem cells (hNSCs), by which TNFα possibly fulfills important roles in neuronal impairment. The results show that PPARγ mediates the cell viability of hNSCs via the downregulation of the activity of caspase 3, indicating that this rescue effect of PPARγ could improve the reduced levels of two mitochondrial regulators, adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1) in the hNSCs with TNFα. The stimulation of mitochondrial function by PPARγ was associated with activation of the PPAR coactivator1 alpha (PGC1α) pathway by up-regulation of oxidative defense and mitochondrial systems. The above protective effects appeared to be exerted by a direct activation of the rosiglitazone, because it protected hNSCs from TNFα-evoked oxidative stress and mitochondrial deficiency. Here we show that the rosiglitazone protects hNSCs against Aβ-induced apoptosis and promotes cell survival. These findings extend our understanding of the central role of PPARγ in TNFα-related neuronal impairment, which probably increases risks of neurodegenerative diseases. The anti-inflammatory effects of PPARγ in the hNSCs with TNFα, and the involved mechanisms were also characterized.

Rosiglitazone activation of PPARγ-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced mitochondrial dysfunction and oxidative stress.

Neuronal cell impairment, such as that induced by amyloid-beta (Aβ) protein, is a process with limited therapeutic interventions and often leads to long-term neurodegeneration common in disorders such as Alzheimers disease. Interestingly, peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor whose ligands control many physiological and pathologic processes, and may be neuroprotective. We hypothesized that rosiglitazone, a PPARγ agonist, would prevent Aβ-mediated effects in human neural stem cells (hNSCs). Here, we show that rosiglitazone reverses, via PPARγ-dependent downregulation of caspase 3 and 9 activity, the Aβ-mediated decreases in hNSC cell viability. In addition, Aβ decreases hNSC messenger RNA (mRNA) levels of 2 neuroprotective factors (Bcl-2 and CREB), but co-treatment with rosiglitazone significantly rescues these effects. Rosiglitazone co-treated hNSCs also showed significantly increased mitochondrial function (reflected by levels of adenosine triphosphate and Mit mass), and PPARγ-dependent mRNA upregulation of PGC1α and mitochondrial genes (nuclear respiratory factor-1 and Tfam). Furthermore, hNSCs co-treated with rosiglitazone were significantly rescued from Aβ-induced oxidative stress and correlates with reversal of the Aβ-induced mRNA decrease in oxidative defense genes (superoxide dismutase 1, superoxide dismutase 2, and glutathione peroxidase 1). Taken together, these novel findings show that rosiglitazone-induced activation of PPARγ-dependent signaling rescues Aβ-mediated toxicity in hNSCs and provide evidence supporting a neuroprotective role for PPARγ activating drugs in Aβ-related diseases such as Alzheimers disease.

Rosiglitazone promotes neurite outgrowth and mitochondrial function in N2A cells via PPARgamma pathway.

Several pieces of evidence indicate that peroxisome proliferator-activated receptor gamma (PPARγ) stimulation promotes neuronal differentiation. However, to date, the effects of a synthetic PPARγ agonist (Rosiglitazone, Rosi) on neurite outgrowth have not yet been well described. Here we have evaluated the effects of Rosi on neurite outgrowth and mitochondrial function in the mouse neuroblastoma Neuro 2a (N2A) cell line. Our results show that Rosi promotes neurite outgrowth of N2A cells and significantly increases the population of neurite-bearing cells, with apparent increase of intracellular calcium and the expression of calmodulin-dependent kinase I (CaMKI). Rosi also increases the intracellular cAMP and expression of both protein kinase A (PKA) and cAMP response element binding protein (CREB). Phosphorylation of CREB was also detected in the Rosi treated N2A cells. Moreover, Rosi significantly increases the transcription of AMP-activated kinase (AMPK) and Sirtuin 1 (SIRT1). Besides, the expression of PPAR coactivator 1α (PGC1α), as well as the mRNA level its downstream genes, including nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam) were induced by Rosi treatments. Furthermore, Rosi increases the level of ATP, D-loop, and mitochondrial mass in N2A cells. Collectively, these findings provide an array of evidence that PPARγ activation provides beneficial neuronal networks within neurite outgrowth.

Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction

Alzheimers disease (AD) is the general consequence of dementia and is diagnostic neuropathology by the cumulation of amyloid-beta (Aβ) protein aggregates, which are thought to promote mitochondrial dysfunction processes leading to neurodegeneration. AMP-activated protein kinase (AMPK), a critical regulator of energy homeostasis and a major player in lipid and glucose metabolism, is potentially implied in the mitochondrial deficiency of AD. Metformin, one of the widespread used anti- metabolic disease drugs, use its actions in part by stimulation of AMPK. While the mechanisms of AD are well established, the neuronal roles for AMPK in AD are still not well understood. In the present study, human neural stem cells (hNSCs) exposed to Aβ had significantly reduced cell viability, which correlated with decreased AMPK, neuroprotective genes (Bcl-2 and CREB) and mitochondria associated genes (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3/9 activity and cytosolic cytochrome c. Co-treatment with metformin distinct abolished the Aβ-caused actions in hNSCs. Metformin also significantly rescued hNSCs from Aβ-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Importantly, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology in the hNSCs with Aβ. These findings extend our understanding of the central role of AMPK in Aβ-related neuronal impairment. Thus, a better understanding of AMPK might assist in both the recognition of its critical effects and the implementation of new therapeutic strategies in the treatment of AD.

Rosiglitazone activation of PPARγ-dependent signaling is neuroprotective in mutant huntingtin expressing cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a crucial transcription factor for neuroprotection in several brain diseases. Using a mouse model of Huntingtons Disease (HD), we recently showed that PPARγ not only played a major function in preventing HD, but also oral intake of a PPARγ agonist (thiazolidinedione, TZD) significantly reduced the formation of mutant Huntingtin (mHtt) aggregates in the brain (e.g., cortex and striatum). The molecular mechanisms by which PPARγ exerts its HD neuroprotective effects remain unresolved. We investigated whether the PPARγ agonist (rosiglitazone) mediates neuroprotection in the mHtt expressing neuroblastoma cell line (N2A). Here we show that rosiglitazone upregulated the endogenous expression of PPARγ, its downstream target genes (including PGC1α, NRF-1 and Tfam) and mitochondrial function in mHtt expressing N2A cells. Rosiglitazone treatment also significantly reduced mHtt aggregates that included ubiquitin (Ub) and heat shock factor 1 (HSF1), as assessed by a filter-retardation assay, and increased the levels of the functional ubiquitin-proteasome system (UPS), HSF1 and heat shock protein 27/70 (HSP27/70) in N2A cells. Moreover, rosiglitazone treatment normalized endoplasmic reticulum (ER) stress sensors Bip, CHOP and ASK1, and significantly increased N2A cell survival. Taken together, these findings unveil new insights into the mechanisms by which activation of PPARγ signaling protects against the HD-mediated neuronal impairment. Further, our data also support the concept that PPARγ may be a novel therapeutic target for treating HD.

Akt1 mediates neuronal differentiation in zebrafish via a reciprocal interaction with notch signaling.

Akt1 is well known for its role in regulating cell proliferation, differentiation, and apoptosis and is implicated in tumors and several neurological disorders. However, the role of Akt1 in neural development has not been well defined. We have isolated zebrafish akt1 and shown that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. Injection of akt1 morpholinos resulted in loss of neuronal precursors with a concomitant increase in post-mitotic neurons, indicating that knockdown of Akt1 is sufficient to cause premature differentiation of neurons. A similar phenotype was observed in embryos deficient for Notch signaling. Both the ligand (deltaA) and the downstream target of Notch (her8a) were downregulated in akt1 morphants, indicating that Akt1 is required for Delta-Notch signaling. Furthermore, akt1 expression was downregulated in Delta-Notch signaling-deficient embryos and could be induced by constitutive activation of Notch signaling. In addition, knockdown of Akt1 was able to nullify the inhibition of neuronal differentiation caused by constitutive activation of Notch signaling. Taken together, these results provide in vivo evidence that Akt1 interacts with Notch signaling reciprocally and provide an explanation of why Akt1 is essential for the inhibition of neuronal differentiation.

Resveratrol activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced inflammation and oxidative stress

ABSTRACT Alzheimers disease (AD) is a neurodegenerative disorder with progressive memory loss resulting in dementia. Amyloid‐beta (A&bgr;) peptides play a critical role in the pathogenesis of this disease, and are thought to promote inflammation and oxidative stress leading to neurodegeneration in the neocortex and hippocampus of the AD brains. AMP‐activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis, and cell survival in response to inflammation and oxidative stress. However, the neuroprotective mechanisms by which AMPK achieves these beneficial effects in human neural stem cells (hNSCs) exposed to A&bgr; is still not well understood. Resveratrol is a potent activator of AMPK suggesting it may have therapeutic potential against AD. Therefore, we will test the hypothesis that the AMPK activator resveratrol protects against A&bgr; mediated neuronal impairment (inflammation and oxidative stress) in hNSCs. Here, A&bgr;‐treated hNSCs had significantly decreased cell viability that correlated with increased TNF‐&agr; and IL‐1&bgr; inflammatory cytokine expression. Co‐treatment with resveratrol significantly abrogated the A&bgr;‐mediated effects in hNSCs, and was effectively blocked by the addition of the AMPK‐specific antagonist (Compound C). These results suggest the neuroprotective effects of resveratrol are mediated by an AMPK‐dependent pathway. In addition, resveratrol rescued the transcript expression levels of inhibitory kappa B kinase (IKK) in A&bgr;‐treated hNSCs. NF‐&kgr;B is a transcription factor with a key role in the expression of a variety of genes involved in inflammatory responses. Resveratrol prevented the A&bgr;‐mediated increases in NF‐&kgr;B mRNA and protein levels, and its nuclear translocation in hNSCs. Co‐treatment with resveratrol also significantly restored iNOS and COX‐2 levels in A&bgr;‐treated hNSCs. Furthermore, hNSCs co‐treated with resveratrol were significantly rescued from A&bgr;‐induced oxidative stress, which correlated with reversal of the A&bgr;‐induced mRNA decrease in oxidative defense genes (SOD‐1, NRF2, Gpx1, Catalase, GSH and HO‐1). Taken together, these novel findings show that activation of AMPK‐dependent signaling by resveratrol rescues A&bgr;‐mediated neurotoxicity in hNSCs, and provides evidence supporting a neuroprotective role for AMPK activating drugs in A&bgr;‐related diseases such as AD. HighlightsResveratrol rescues viability, TNF&agr; and IL‐1&bgr; in A&bgr;‐treated hNSCs.Resveratrol improves inflammatory responses and genes in A&bgr;‐treated hNSCs.Resveratrol normalizes oxidative stress and defense genes in A&bgr;‐treated hNSCs.

Metformin activation of AMPK suppresses AGE-induced inflammatory response in hNSCs

ABSTRACT A growing body of evidence suggests type 2 diabetes mellitus (T2DM) is linked to neurodegenerative diseases such as Alzheimers disease (AD). Although the precise mechanisms remain unclear, T2DM may exacerbate neurodegenerative processes. AMP‐activated protein kinase (AMPK) signaling is an evolutionary preserved pathway that is important during homeostatic energy biogenesis responses at both the cellular and whole‐body levels. Metformin, a ubiquitously prescribed anti‐diabetic drug, exerts its effects by AMPK activation. However, while the roles of AMPK as a metabolic mediator are generally well understood, its performance in neuroprotection and neurodegeneration are not yet well defined. Given hyperglycemia is accompanied by an accelerated rate of advanced glycosylation end product (AGE) formation, which is associated with the pathogenesis of diabetic neuronal impairment and, inflammatory response, clarification of the role of AMPK signaling in these processes is needed. Therefore, we tested the hypothesis that metformin, an AMPK activator, protects against diabetic AGE induced neuronal impairment in human neural stem cells (hNSCs). In the present study, hNSCs exposed to AGE had significantly reduced cell viability, which correlated with elevated inflammatory cytokine expression, such as IL‐1&agr;, IL‐1&bgr;, IL‐2, IL‐6, IL‐12 and TNF‐&agr;. Co‐treatment with metformin significantly abrogated the AGE‐mediated effects in hNSCs. In addition, metformin rescued the transcript and protein expression levels of acetyl‐CoA carboxylase (ACC) and inhibitory kappa B kinase (IKK) in AGE‐treated hNSCs. NF‐&kgr;B is a transcription factor with a key role in the expression of a variety of genes involved in inflammatory responses, and metformin did prevent the AGE‐mediated increase in NF‐&kgr;B mRNA and protein levels in the hNSCs exposed to AGE. Indeed, co‐treatment with metformin significantly restored inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) levels in AGE‐treated hNSCs. These findings extend our understanding of the central role of AMPK in AGE induced inflammatory responses, which increase the risk of neurodegeneration in diabetic patients.

The neuroprotective role of rosiglitazone in advanced glycation end product treated human neural stem cells is PPARgamma-dependent

Hyperglycemia is accompanied by an accelerated formation rate of advanced glycation end products (AGEs), which is associated with the pathogenesis of diabetic neuronal deficits. Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors and its ligands are known to control many physiological, pathological and inflammatory pathways. Weinvestigated the hypothesis that the PPARγ agonist (rosiglitazone) would abrogate AGEs-mediated neurotoxic effects on human neural stem cells (hNSCs), by whichAGEs may play a role in diabetic-related neuronal impairment. Here, we show that rosiglitazone treatment increases cell viability of hNSCs via downregulation of caspase 3 activity. These rescue effects were extended in our studies showingrosiglitazone-mediated activation of PPARγ reversed the expression levels of two neuroprotective factors (Bcl-2 and PGC1α) that were downregulated in hNSCs exposed to AGEs alone. The stimulation of mitochondrial function and anti-oxidative stress by rosiglitazone was associated with activation of the PGC1α pathway by up-regulation of mitochondrial (NRF-1 and Tfam) and oxidative defense (SOD1, SOD2 and Gpx1) genes. Moreover, rosiglitazone significantly normalized the inflammatory responses (TNF-α and IL-1β), NF-κB (p65), and inflammatory genes (iNOS and COX-2) in the hNSCs treated with AGEs. This neuroprotective effect of rosiglitazone was effectively blocked by PPARγ-specific antagonist (GW9662), demonstrating that the action of rosiglitazone was mediated by at PPARγ-dependent pathway. Collectively, these novel findings show AGEs induce neurotoxic effects in hNSCs, and provide important mechanistic insights that may explain the increased risk of neuronal impairment deficits in diabetic patients. More importantly, these data show rosiglitazone-mediated activation of PPARγ-dependent signaling is neuroprotective in AGE-treated hNSCs, and suggests PPARγ ligands may be useful in the therapeutic management of patients with neurodegenerative diseases.