Nilotinib Improves Bioenergetic Profiling in Brain Astroglia in the 3xTg Mouse Model of Alzheimer's Disease.

Current treatments targeting amyloid beta in Alzheimer's disease (AD) have minimal efficacy, which results in a huge unmet medical need worldwide. Accumulating data suggest that brain mitochondrial dysfunction play a critical role in AD pathogenesis. Targeting cellular mechanisms associated with mitochondrial dysfunction in AD create a novel approach for drug development. This study investigated the effects of nilotinib, as a selective tyrosine kinase inhibitor, in astroglia derived from 3xTg-AD mice versus their C57BL/6-controls. Parameters included oxygen consumption rates (OCR), ATP, cytochrome c oxidase (COX), citrate synthase (CS) activity, alterations in oxidative phosphorylation (OXPHOS), nuclear factor kappa B (NF-κB), key regulators of mitochondrial dynamics (mitofusin (Mfn1), dynamin-related protein 1 (Drp1)), and mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α), calcium/calmodulin-dependent protein kinase II (CaMKII), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)). Nilotinib increased OCR, ATP, COX, Mfn1, and OXPHOS levels in 3xTg astroglia. No significant differences were detected in levels of Drp1 protein and CS activity. Nilotinib enhanced mitochondrial numbers, potentially through a CaMKII-PGC1α-Nrf2 pathway in 3xTg astroglia. Additionally, nilotinib-induced OCR increases were reduced in the presence of the NF-κB inhibitor, Bay11-7082. The data suggest that NF-κB signaling is intimately involved in nilotinib-induced changes in bioenergetics in 3xTg brain astroglia. Nilotinib increased translocation of the NF-κB p50 subunit into the nucleus of 3xTg astroglia that correlates with an increased expression and activation of NF-κB. The current findings support a role for nilotinib in improving mitochondrial function and suggest that astroglia may be a key therapeutic target in treating AD.

Deciphering the neuroprotective and neurogenic potential of soluble amyloid precursor protein alpha (sAPPα).

Amyloid precursor protein (APP) is a transmembrane protein expressed largely within the central nervous system. Upon cleavage, it does not produce the toxic amyloid peptide (Aß) only, which is involved in neurodegenerative progressions but via a non-amyloidogenic pathway it is metabolized to produce a soluble fragment (sAPPα) through α-secretase. While a lot of studies are focusing on the role played by APP in the pathogenesis of Alzheimer's disease, sAPPα is reported to have numerous neuroprotective effects and it is being suggested as a candidate with possible therapeutic potential against Alzheimer's disease. However, the mechanisms through which sAPPα precisely works remain elusive. We have presented a comprehensive review of how sAPPα is regulating the neuroprotective effects in different biological models. Moreover, we have focused on the role of sAPPα during different developmental stages of the brain, neurogenic microenvironment in the brain and how this metabolite of APP is regulating the neurogenesis which is regarded as a compelling approach to ameliorate the impaired learning and memory deficits in dementia and diseases like Alzheimer's disease. sAPPα exerts beneficial physiological, biochemical and behavioral effects mitigating the detrimental effects of neurotoxic compounds. It has shown to increase the proliferation rate of numerous cell types and promised the synaptogenesis, neurite outgrowth, cell survival and cell adhesion. Taken together, we believe that further studies are warranted to investigate the exact mechanism of action so that sAPPα could be developed as a novel therapeutic target against neuronal deficits.

Secreted amyloid precursor protein alpha activates neuronal insulin receptors and prevents diabetes-induced encephalopathy.

Secreted amyloid precursor protein alpha (sAPPα) is a potent neurotrophin in the CNS but a dedicated receptor has not been found. However, protein interactions involving amyloid beta (Aß), a peptide cleaved from the same parent peptide as sAPPα, indicate that insulin receptors (IRs) could be a target of amyloid peptides. In this study, in vitro analysis of cortical neuronal cultures revealed that exogenous sAPPα increased IR phosphorylation in the absence of insulin. Furthermore, in an APP overexpressing mouse model, sAPPα bound IRs in the cortex with significantly greater binding in hypoinsulinemic animals. To further examine the effects of sAPPα on the diabetic brain, we next rendered sAPPα overexpressing mice insulin depleted and found that sAPPα blocked aberrant tau phosphorylation (T231) in cortical tissue after 16 weeks diabetes. sAPPα overexpression also prevented hyperphosphorylation of AKT/GSK3 and activation of the unfolded protein response (UPR). In total, these data show sAPPα binds and activates neuronal IRs and that sAPPα has a protective effect on diabetic brain tissue.

Early growth response 2 (Egr-2) expression is triggered by NF-κB activation.

Transcription factors are known to play multiple roles in cellular function. Investigators report that factors such as early growth response (Egr) protein and nuclear factor kappa B (NF-κB) are activated in the brain during cancer, brain injury, inflammation, and/or memory. To explore NF-κB activity further, we investigated the transcriptomes of hippocampal slices following electrical stimulation of NF-κB p50 subunit knockout mice (p50-/-) versus their controls (p50+/+). We found that the early growth response gene Egr-2 was upregulated by NF-κB activation, but only in p50+/+ hippocampal slices. We then stimulated HeLa cells and primary cortical neurons with tumor necrosis factor alpha (TNFα) to activate NF-κB and increase the expression of Egr-2. The Egr-2 promoter sequence was analyzed for NF-κB binding sites and chromatin immunoprecipitation (ChIP) assays were performed to confirm promoter occupancy in vivo. We discovered that NF-κB specifically binds to an NF-κB consensus binding site within the proximal promoter region of Egr-2. Luciferase assay demonstrated that p50 was able to transactivate the Egr-2 promoter in vitro. Small interfering RNA (siRNA)-mediated p50 knockdown corroborated other Egr-2 expression studies. We show for the first time a novel link between NF-κB activation and Egr-2 expression with Egr-2 expression directly controlled by the transcriptional activity of NF-κB.

Temporal dystrophic remodeling within the intrinsic cardiac nervous system of the streptozotocin-induced diabetic rat model.

INTRODUCTION: The pathogenesis of heart failure (HF) in diabetic individuals, called "diabetic cardiomyopathy", is only partially understood. Alterations in the cardiac autonomic nervous system due to oxidative stress have been implicated. The intrinsic cardiac nervous system (ICNS) is an important regulatory pathway of cardiac autonomic function, however, little is known about the alterations that occur in the ICNS in diabetes. We sought to characterize morphologic changes and the role of oxidative stress within the ICNS of diabetic hearts. Cultured ICNS neuronal cells from the hearts of 3- and 6-month old type 1 diabetic streptozotocin (STZ)-induced diabetic Sprague-Dawley rats and age-matched controls were examined. Confocal microscopy analysis for protein gene product 9.5 (PGP 9.5) and amino acid adducts of (E)-4-hydroxy-2-nonenal (4-HNE) using immunofluorescence was undertaken. Cell morphology was then analyzed in a blinded fashion for features of neuronal dystrophy and the presence of 4-HNE adducts. RESULTS: At 3-months, diabetic ICNS neuronal cells exhibited 30% more neurite swellings per area (p = 0.01), and had a higher proportion with dystrophic appearance (88.1% vs. 50.5%; p = <0.0001), as compared to control neurons. At 6-months, diabetic ICNS neurons exhibited more features of dystrophy as compared to controls (74.3% vs. 62.2%; p = 0.0448), with 50% more neurite branching (p = 0.0015) and 50% less neurite outgrowth (p = <0.001). Analysis of 4-HNE adducts in ICNS neurons of 6-month diabetic rats demonstrated twice the amount of reactive oxygen species (ROS) as compared to controls (p = <0.001). CONCLUSION: Neuronal dystrophy occurs in the ICNS neurons of STZ-induced diabetic rats, and accumulates temporally within the disease process. In addition, findings implicate an increase in ROS within the neuronal processes of ICNS neurons of diabetic rats suggesting an association between oxidative stress and the development of dystrophy in cardiac autonomic neurons.

Strain specific differences in memory and neuropathology in a mouse model of Alzheimer's disease.

AIMS: Studies using transgenic mouse strains that incorporate Alzheimer's disease (AD) mutations are valuable for the identification of signaling pathways, potential drug targets, and possible mechanisms of disease that will aid in our understanding of AD. However, reports on the effects of specific AD mutations (Swedish, KM670/671NL; Indiana, V717F) on behavior (Morris water maze) and neuropathological progression have been inconsistent when comparing different genetic backgrounds in these models. Given this, investigators are compelled to more closely evaluate different background strains. The aim of the present study was to compare two commonly used TgCRND8 backgrounds, the 129SvEvTac/C57F1 strain and the C3H/C57F1 strain. MAIN METHODS: Memory function was assessed by the Morris water maze, a test for assaying hippocampal-dependent memory. We also stained with ThioflavinS in order to visualize and quantify amyloid beta (Abeta) plaques. Real time polymerase chain reaction (PCR) was used to measure insulin-degrading enzyme (IDE), an enzyme that also degrades amyloid beta. KEY FINDINGS: We found deficits in the 129SvEvTac/C57F1 strain in several parameters of the Morris water maze. In addition, amyloid plaque load expression was significantly greater in the 129SvEvTac/C57F1 as compared to the C3H/C57F1 strain as demonstrated by histochemical staining. We also observed a significant decrease in IDE, in the 129SvEvTac/C57F1 strain. SIGNIFICANCE: This study supports the notion that strain specific differences are apparent in tests of spatial memory and neuropathologic progression in AD.

Nuclear factor-kappaB activation in axons and Schwann cells in experimental sciatic nerve injury and its role in modulating axon regeneration: studies with etanercept.

Early inflammatory events may inhibit functional recovery after injury in both the peripheral and central nervous systems. We investigated the role of the inflammatory tumor necrosis factor/nuclear factor-kappaB (NF-kappaB) axis on events subsequent to sciatic nerve crush injury in adult rats. Electrophoretic mobility shift assays revealed that within 6 hours after crush, NF-kappaB DNA-binding activity increased significantly in a 1-cm section around the crush site. By immunofluorescence staining, there was increased nuclear localization of the NF-kappaB subunits p50 but not p65 or c-Rel in Schwann cells but no obvious inflammatory cell infiltration. In rats injected subcutaneously with etanercept, a tumor necrosis factor receptor chimera that binds free cytokine, the injury-induced rise in NF-kappaB DNA-binding activity was inhibited, and nuclear localization of p50 in Schwann cells was lowered after the injury. Axonal growth 3 days after nerve crush assessed with immunofluorescence for GAP43 demonstrated that the regeneration distance of leading axons from the site of nerve crush was greater in etanercept-treated animals than in saline-treated controls. These data indicate that tumor necrosis factor mediates rapid activation of injury-induced NF-kappaB DNA binding in Schwann cells and that these events are associated with inhibition of postinjury axonal sprouting.

Neuregulin beta1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neurons.

Modulation of intracellular free calcium levels is the primary second messenger system of the neuronal glutamatergic system, playing a role in regulation of all major cellular processes. The protein neuregulin (NRG) beta1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic glutamate receptors. The effect NRG may have on glutamate-induced changes in intracellular free calcium concentrations has not been examined, however. In this study, cultured embryonic rat hippocampal neurons were treated with NRGbeta1 to determine a possible effect on glutamate-induced intracellular calcium levels. Long-term (24 h), but not short-term (1 h), incubation with NRGbeta1 resulted in a significantly greater glutamate-mediated acute peak elevation of intracellular calcium levels than occurred in vehicle-treated neurons. Long-term NRGbeta1 incubation significantly enhanced calcium increase induced by specific stimulation of metabotropic glutamate receptors, but did not significantly alter the N-methyl D-aspartate (NMDA)- or KCl-induced calcium increase and paradoxically decreased the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) treatment on intracellular calcium. Metabotropic glutamate receptors cause increased intracellular free calcium via release of calcium from intracellular stores; thus this system was examined in more detail. NRGbeta1 treatment significantly (greater than 2-fold) enhanced calcium release from endoplasmic reticulum stores after stimulation of ryanodine receptors with caffeine, but did not significantly increase calcium release from endoplasmic reticulum mediated by inositol trisphosphate (IP3) receptors. In addition, ryanodine receptor inhibition with ruthenium red prevented the glutamate-induced increase in intracellular calcium levels in NRGbeta1-treated neurons. These data show that long-term NRGbeta1 treatment can enhance glutamate-induced peak intracellular calcium levels through metabotropic glutamate receptor activation by increasing endoplasmic reticulum calcium release through ryanodine receptors.

Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.

Insulin deficiency in type I diabetes may lead to cognitive impairment, cerebral atrophy and white matter abnormalities. We studied the impact of a novel delivery system using intranasal insulin (I-I) in a mouse model of type I diabetes (streptozotocin-induced) for direct targeting of pathological and cognitive deficits while avoiding potential adverse systemic effects. Daily I-I, subcutaneous insulin (S-I) or placebo in separate cohorts of diabetic and non-diabetic CD1 mice were delivered over 8 months of life. Radio-labelled insulin delivery revealed that I-I delivered more rapid and substantial insulin levels within the cerebrum with less systemic insulin detection when compared with S-I. I-I delivery slowed development of cognitive decline within weekly cognitive/behavioural testing, ameliorated monthly magnetic resonance imaging abnormalities, prevented quantitative morphological abnormalities in cerebrum, improved mouse mortality and reversed diabetes-mediated declines in mRNA and protein for phosphoinositide 3-kinase (PI3K)/Akt and for protein levels of the transcription factors cyclic AMP response element binding protein (CREB) and glycogen synthase kinase 3beta (GSK-3beta) within different cerebral regions. Although the murine diabetic brain was not subject to cellular loss, a diabetes-mediated loss of protein and mRNA for the synaptic elements synaptophysin and choline acetyltransferase was prevented with I-I delivery. As a mechanism of delivery, I-I accesses the brain readily and slows the development of diabetes-induced brain changes as compared to S-I delivery. This therapy and delivery mode, available in humans, may be of clinical utility for the prevention of pathological changes in the diabetic human brain.

Distal degenerative sensory neuropathy in a long-term type 2 diabetes rat model.

OBJECTIVE: Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats. RESEARCH DESIGN AND METHODS: We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons. RESULTS: ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor beta-subunit (IRbeta), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IRbeta protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the alpha2delta1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-kappaB (NF-kappaB; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-kappaB, and RAGE protein. CONCLUSIONS: ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.

TGF-beta1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis.

Alzheimer's disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimer's disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.

NF-kappaB activated by ER calcium release inhibits Abeta-mediated expression of CHOP protein: enhancement by AD-linked mutant presenilin 1.

Mutations in presenilin which result in early-onset Alzheimer disease (AD) cause both increased calcium release from intracellular stores, primarily endoplasmic reticulum (ER), and changes in NF-kappaB activation. Some studies have also reported that neurons containing AD-linked mutant presenilins (mPS1) show increased vulnerability to various stresses, while others report no differences in neuronal death. The majority of these reports center on potential changes in ER stress, because of the enhanced ER calcium release seen in mPS1 neurons. One of the primary death effectors of ER stress is CHOP, also termed GADD153, which acts to transcriptionally inhibit protective cellular molecules such as Bcl-2 and glutathione. Because both CHOP and NF-kappaB are activated by increased intracellular calcium and stress, yet have diametrically opposite effects on neuronal vulnerability, we sought to examine this interaction in greater detail. We observed that IP3-mediated calcium release from ER, stimulated by Abeta exposure, mediated both CHOP expression and NF-kappaB DNA binding activity. Further, specific inhibition of NF-kappaB resulted in greater expression of CHOP, while activation of NF-kappaB inhibited CHOP expression. The enhanced release of calcium from IP3-mediated ER stores in mPS1 neurons stimulated increased NF-kappaB compared to normal neurons, which inhibited CHOP expression. Upon blockage of NF-kappaB, exposure to Abeta caused significantly greater Abeta-mediated CHOP expression and death in mPS1 neurons compared to normal neurons. Thus, AD-linked PS1 mutations disrupt the balance between stress-induced NF-kappaB and CHOP, resulting in greater dependence on stress-induced NF-kappaB activation in mPS1 neurons.

Activation of nuclear factor-kappaB via endogenous tumor necrosis factor alpha regulates survival of axotomized adult sensory neurons.

Embryonic dorsal root ganglion (DRG) neurons die after axonal damage in vivo, and cultured embryonic DRG neurons require exogenous neurotrophic factors that activate the neuroprotective transcription factor nuclear factor-kappaB (NF-kappaB) for survival. In contrast, adult DRG neurons survive permanent axotomy in vivo and in defined culture media devoid of exogenous neurotrophic factors in vitro. Peripheral axotomy in adult rats induces local accumulation of the cytokine tumor necrosis factor alpha (TNFalpha), a potent activator of NF-kappaB activity. We tested the hypothesis that activation of NF-kappaB stimulated by endogenous TNFalpha was required for survival of axotomized adult sensory neurons. Peripheral axotomy of lumbar DRG neurons by sciatic nerve crush induced a very rapid (within 2 h) and significant elevation in NF-kappaB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-kappaB nuclear translocation and a significant rise in NF-kappaB DNA-binding activity after plating. Inhibitors of NF-kappaB (SN50 or NF-kappaB decoy DNA) resulted in necrotic cell death of medium to large neurons (> or =40 microm) within 24 h (60 and 75%, respectively), whereas inhibition of p38 and mitogen-activated protein/extracellular signal-regulated kinase did not effect survival. ELISA revealed that these cultures contained TNFalpha, and exposure to an anti-TNFalpha antibody inhibited NF-kappaB DNA-binding activity by approximately 35% and killed approximately 40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-kappaB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.