Christopher J. Nicol

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (−) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

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.

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 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.

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ(+/−) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.

Stromal Adipocyte PPARγ Protects Against Breast Tumorigenesis

Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.

The Key to Unlocking the Chemotherapeutic Potential of PPARγ Ligands: Having the Right Combination.

Despite extensive preclinical evidence that peroxisome proliferator-activated receptor (PPAR)γ activation protects against tumourigenesis, results from a few clinical trials using PPARγ ligands as monotherapy show modest success. In spite of this, several groups reported exciting results with therapeutic regimens that combine PPARγ ligands with other compounds: chemotherapeutic agents, retinoid x receptor (RXR)α agonists, statins, or cell-to-cell signaling molecules in preclinical cancer models and human trials. Here we have compiled an extensive review, consolidating the existing literature, which overwhelmingly supports a beneficial effect of treating with PPARγ ligands in combination with existing chemotherapies versus their monotherapy in cancer. There are many examples in which combination therapy resulted in synergistic/additive effects on apoptosis, differentiation, and the ability to reduce cell growth and tumour burden. There are also studies that indicate that PPARγ ligand pretreatment overcomes resistance and reduces toxicities. Several mechanisms are explored to explain these protective effects. This paper highlights each of these studies that, collectively, make a very strong case for the use of PPARγ ligands in combination with other agents in the treatment and management of several cancers.

PPAR Medicines and Human Disease: The ABCs of It All

ATP-dependent binding cassette (ABC) transporters are a family of transmembrane proteins that pump a variety of hydrophobic compounds across cellular and subcellular barriers and are implicated in human diseases such as cancer and atherosclerosis. Inhibition of ABC transporter activity showed promise in early preclinical studies; however, the outcomes in clinical trials with these agents have not been as encouraging. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate genes involved in fat and glucose metabolism, and inflammation. Activation of PPAR signaling is also reported to regulate ABC gene expression. This suggests the potential of PPAR medicines as a novel means of controlling ABC transporter activity at the transcriptional level. This paper summarizes the advances made in understanding how PPAR medicines affect ABC transporters, and the potential implications for impacting on human diseases, in particular with respect to cancer and atherosclerosis.

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.