Altered expression of IGF‐I system in neurons of the inflamed spinal cord during acute experimental autoimmune encephalomyelitis

There is growing evidence that the impaired IGF‐I system contributes to neurodegeneration. In this study, we examined the spinal cords of the EAE, the animal model of multiple sclerosis, to see if the expression of the IGF‐I system is altered. To induce EAE, C57/BL6 mice were immunized with the Hooke lab MOG kit, sacrificed at the peak of the disease and their spinal cords were examined for the immunoreactivities (ir) of the IGF‐I, IGF binding protein‐1 (IGFBP‐1) and glycogen synthase kinase 3β (GSK3β), as one major downstream molecule in the IGF‐I signaling. Although neurons in the non EAE spinal cords did not show the IGF‐I immunoreactivity, they were numerously positive for the IGFBP‐1. In the inflamed EAE spinal cord however, the patterns of expressions were reversed, that is, a significant increased number of IGF‐I expressing neurons versus a reduced number of IGFBP‐1 positive neurons. Moreover, while nearly all IGF‐I‐ir neurons expressed GSK3β, some expressed it more intensely. Considering our previous finding where we showed a significant reduced number of the inactive (phosphorylated) but not that of the total GSK3β expressing neurons in the EAE spinal cord, it is conceivable that the intense total GSK3β expression in the IGF‐I‐ir neurons belongs to the active form of GSK3β known to exert neuroinflammatory effects. We therefore suggest that the altered expression of the IGF‐I system including GSK3β in spinal cord neurons might involve in pathophysiological events during the EAE.     

β‐catenin mediates insulin‐like growth factor‐I actions to promote cyclin D1 mRNA expression,cell proliferation and survival in oligodendroglial cultures

By promoting cell proliferation, survival and maturation insulin‐like growth factor (IGF)‐I is essential to the normal growth and development of the central nervous system. It is clear that IGF‐I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)‐Akt kinases and MAP kinases signal many of IGF‐I‐IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether β‐catenin, a molecule that is a downstream target of glycogen synthase kinase‐3β (GSK3β) and plays a key role in the Wnt canonical signaling pathway, mediates IGF‐I actions. We found that IGF‐I increases β‐catenin protein abundance within an hour after IGF‐I‐induced phosphorylation of Akt and GSK3β. Inhibiting the PI3‐Akt pathway suppressed IGF‐I‐induced increases in β‐catenin and cyclin D1 mRNA, while suppression of GSK3β activity simulated IGF‐I actions. Knocking‐down β‐catenin mRNA by RNA interference suppressed IGF‐I‐stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that β‐catenin is an important downstream molecule in the PI3‐Akt‐GSK3β pathway, and as such it mediates IGF‐I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. © 2010 Wiley‐Liss, Inc.     

Increased oxidative stress in patients with amyotrophic lateral sclerosis/Parkinsonism‐dementia complex in the Kii peninsula,Japan

Amyotrophic lateral sclerosis and Parkinsonism‐dementia complex of the Kii peninsula (Kii ALS/PDC) is an endemic and a tauopathy, which shows clinical symptoms of amyotrophy, parkinsonism, and dementia. The objective of this study was to report the role of oxidative stress on Kii ALS/PDC using biochemical analysis. Urinary 8‐hydroxydeoxyguanosine (8‐OHdG)/creatinine ratio was analyzed in 11 patients with Kii ALS/PDC and 8 normal controls. The mean level of urinary 8‐OHdG/creatinine ratio of the patients with Kii ALS/PDC was significantly higher than that of control subjects. Oxidative stress may be implicated in pathogenesis of Kii ALS/PDC. © 2008 Movement Disorder Society     

ALS in the Kii Peninsula of Japan,with Special Reference to Neurofibrillary Tangles and Aluminum

Epidemiological and environmental findings of the foci of amyotrophi lateral sclerosis (ALS) in the Western Pacific indicate a possible contributing role of metals, especially aluminum, in the pathogenesis. Al is also a candidate as a causative agent in the formation of neurofibrillary tangles (NFTs), which are characteristic of Guam ALS. To elucidate the pathogenesis of ALS, we conducted neuropathological examination and metal analysis, with special reference to NFT and Al in cases of ALS in the Kii Peninsula of Japan. We examined six of these ALS cases, ten ALS cases from non-focused areas (non-focused ALS) and eight disease controls. The neuropathologic characteristics of Kii ALS were the appearance of NFT in Ammon's horn, temporal cortex, mid-brain and pons, in ccombination with typical ALS pathology. No senile plaque was found in the Kii cases. Non-foused ALS patients rarely showed NFTs in Ammon's horn. Al analysis of Ammon's horns by particle-induced X-ray emission spectrometry (PIXE) revealed a high level of Al signals in the Kii cases, with positive correlation between Al content and NFT frequency. Kii ALS cases showed high level of Al signals in the cervical and lumbar spinal cords ccompared with non-foused ALS and controls. The Morin fluorescent technique revealed more conspicuous green fluorescence in the cytoplasm and nuclei of both the NFT-laden neurons and the degenerative neurons without NFT in Ammon's horn in the Kii ALS cases than in non-focused ALS cases. Green fluorescence was seen in a small number (2%) of the NFTs themselves. We suggest that Al induces abnormality in cytoskeletal protein such as neurofilaments and/or tau, and may cause the formation of NFT in Ammon's horn and also degeneration of motor neurons in Kii ALS.     

The peripheral messenger RNA expression of glycogen synthase kinase‐3β genes in Alzheimer's disease patients: a preliminary study

Objective: To explore the peripheral leucocytic messenger RNA (mRNA) expression of glycogen synthase kinase‐3β (GSK‐3β) gene in Alzheimer's disease (AD) patients. Methods: Using TaqMan relative quantitative real‐time polymerase chain reaction, we analyzed leucocytic gene expression of GSK‐3β in 48 AD patients and 49 healthy controls. Clinical data of AD patients were also collected. Results: The mRNA expression level of the GSK‐3β gene was significantly higher in the AD group (3.13 ± 0.62) than in the normal group (2.77 ± 0.77). Correlational analyses showed that the mRNA expression level of GSK‐3β gene in AD patients was associated with the age of onset (P= 0.047), age (P= 0.055), and Behavioral Pathology in Alzheimer's Disease Rating Scale total score (P= 0.062) and subscores: aggressiveness score (P= 0.073) and anxieties and phobias score (P= 0.067). Through multivariate regression model, older age, higher anxieties and phobias score and aggressiveness score were associated with higher mRNA expression level of GSK‐3β gene. Conclusion: In AD patients, the mRNA expression level of the GSK‐3β gene is increased and may be related to age and behavioural pathology in AD.     

Differential expression of sPLA2 following spinal cord injury and a functional role for sPLA2‐IIA in mediating oligodendrocyte death

After the initial mechanical insult of spinal cord injury (SCI), secondary mediators propagate a massive loss of oligodendrocytes. We previously showed that following SCI both the total phospholipase activity and cytosolic PLA₂‐IVα protein expression increased. However, the expression of secreted isoforms of PLA₂ (sPLA₂) and their possible roles in oligodendrocyte death following SCI remained unclear. Here we report that mRNAs extracted 15 min, 4 h, 1 day, or 1 month after cervical SCI show marked upregulation of sPLA₂‐IIA and IIE at 4 h after injury. In contrast, SCI induced down regulation of sPLA₂‐X, and no change in sPLA₂‐IB, IIC, V, and XIIA expression. At the lesion site, sPLA₂‐IIA and IIE expression were localized to oligodendrocytes. Recombinant human sPLA₂‐IIA (0.01, 0.1, or 2 μM) induced a dose‐dependent cytotoxicity in differentiated adult oligodendrocyte precursor cells but not primary astrocytes or Schwann cells in vitro. Most importantly, pretreatment with S3319, a sPLA₂‐IIA inhibitor, before a 30 min H₂O₂ injury (1 or 10 mM) significantly reduced oligodendrocyte cell death at 48 h. Similarly, pretreatment with S3319 before injury with IL‐1β and TNFα prevented cell death and loss of oligodendrocyte processes at 72 h. Collectively, these findings suggest that sPLA₂‐IIA and IIE are increased following SCI, that increased sPLA₂‐IIA can be cytotoxic to oligodendrocytes, and that in vitro blockade of sPLA₂ can create sparing of oligodendrocytes in two distinct injury models. Therefore, sPLA₂‐IIA may be an important mediator of oligodendrocyte death and a novel target for therapeutic intervention following SCI. © 2009 Wiley‐Liss, Inc.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

GSK‐3β inhibitors reverse cocaine‐induced synaptic transmission dysfunction in the nucleus accumbens

Nucleus accumbens receives glutamatergic projection from the prefrontal cortex (PFC) and dopaminergic input from the Ventral tegmental area (VTA). Recent studies have suggested a critical role for serine/threonine kinase glycogen synthase kinase 3β (GSK3β) in cocaine‐induced hyperactivity; however, the effect of GSK3β on the modulation of glutamatergic and dopaminergic afferents is unclear. In this study, we found that the GSK3 inhibitors, LiCl (100 mg/kg, i.p.) or SB216763 (2.5 mg/kg, i.p.), blocked the cocaine‐induced hyperlocomotor activity in rats. By employing single‐unit recordings in vivo, we found that pretreatment with either SB216763 or LiCl for 15 min reversed the cocaine‐inhibited firing frequency of medium spiny neuron (MSN) in the nucleus accumbens (NAc). Preperfusion of SB216763 (5 μM) ameliorated the inhibitory effect of cocaine on both the α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (up to 99 ± 6.8% inhibition) and N‐methyl‐D‐aspartic acid receptor (NMDAR)‐mediate EPSC (up to 73 ± 9.7% inhibition) in the NAc in brain slices. The effect of cocaine on AMPA and NMDA receptor‐mediate excitatory postsynaptic current (EPSC) were mimicked by the D₁‐like receptor agonist SKF 38393 and blocked by the D₁‐like receptor antagonist SCH 23390, whereas D₂‐like receptor agonist or antagonist failed to mimic or to block the action of cocaine. Preperfusion of SB216763 for 5 min also ameliorated the inhibitory effect of SKF38393 on both AMPA and NMDA receptor‐mediated components of EPSC, indicate the effect of SB216763 on cocaine was via the D₁‐like receptor. Moreover, cocaine inhibited the presynaptic release of glutamate in the NAc, and SB216763 reversed this effect. In conclusion, D₁ receptor–GSK3β pathway, which mediates glutamatergic transmission in the NAc core through a presynaptic mechanism, plays an important role in acute cocaine‐induced hyperlocomotion.     

Mutant huntingtin and glycogen synthase kinase 3‐β accumulate in neuronal lipid rafts of a presymptomatic knock‐in mouse model of Huntington's disease

Patients with Huntington's disease have an expanded polyglutamine tract in huntingtin and suffer severe brain atrophy and neurodegeneration. Because membrane dysfunction can occur in Huntington's disease, we addressed whether mutant huntingtin in brain and primary neurons is present in lipid rafts, which are cholesterol‐enriched membrane domains that mediate growth and survival signals. Biochemical analysis of detergent‐resistant membranes from brains and primary neurons of wild‐type and presymptomatic Huntington's disease knock‐in mice showed that wild‐type and mutant huntingtin were recovered in lipid raft‐enriched detergent‐resistant membranes. The association with lipid rafts was stronger for mutant huntingtin than wild‐type huntingtin. Lipid rafts extracted from Huntington's disease mice had normal levels of lipid raft markers (G_αq, Ras, and flotillin) but significantly more glycogen synthase kinase 3‐β. Increases in glycogen synthase kinase 3‐β have been associated with apoptotic cell death. Treating Huntington's disease primary neurons with inhibitors of glycogen synthase kinase 3‐β reduced neuronal death. We speculate that accumulation of mutant huntingtin and glycogen synthase kinase 3‐β in lipid rafts of presymptomatic Huntington's disease mouse neurons contributes to neurodegeneration in Huntington's disease. © 2009 Wiley‐Liss, Inc.     

GSK3β overexpression induces neuronal death and a depletion of the neurogenic niches in the dentate gyrus

Overexpression of GSK3β in transgenic mice induces learning deficits and some features associated with Alzheimer's disease (AD), including dentate gyrus (DG) atrophy. Here, we assessed whether these mice also recapitulate DG atrophy as well as impaired neurogenesis reported in AD. Ultrastructural analysis revealed that there were fewer and more disorganized neurogenic niches in these animals, coupled with an increase in the proportion of immature neurons. Indeed, the maturation of granule cells is delayed as witnessed by the alterations to the length and patterning of their dendritic trees and to the mossy fiber terminals. Together with an increase in neuronal death, these phenomena lead to a marked decrease in the number and disorganization of granule cells of the DG. Our results suggest that GSK3β overexpression perturbs proliferation and maturation, resulting in the loss of immature neurons. In turn, the activation of microglia is stimulated in conjunction with a decrease in the birth of new functional neurons, leading to the deterioration of this structure. These data support the idea that by inducing degeneration of the DG, GSK3β could be involved in the pathogenesis of AD. © 2010 Wiley‐Liss, Inc.