Takatoshi Ueki

Methamphetamine Causes Microglial Activation in the Brains of Human Abusers

Methamphetamine is a popular addictive drug whose use is associated with multiple neuropsychiatric adverse events and toxic to the dopaminergic and serotonergic systems of the brain. Methamphetamine-induced neuropathology is associated with increased expression of microglial cells that are thought to participate in either pro-toxic or protective mechanisms in the brain. Although reactive microgliosis has been observed in animal models of methamphetamine neurotoxicity, no study has reported on the status of microglial activation in human methamphetamine abusers. The present study reports on 12 abstinent methamphetamine abusers and 12 age-, gender-, and education-matched control subjects who underwent positron emission tomography using a radiotracer for activated microglia, [11C](R)-(1-[2-chlorophenyl]-N-methyl-N-[1-methylpropyl]-3-isoquinoline carboxamide) ([11C](R)-PK11195). Compartment analysis was used to estimate quantitative levels of binding potentials of [11C](R)-PK11195 in brain regions with dopaminergic and/or serotonergic innervation. The mean levels of [11C](R)-PK11195 binding were higher in methamphetamine abusers than those in control subjects in all brain regions (>250% higher; p < 0.01 for all). In addition, the binding levels in the midbrain, striatum, thalamus, and orbitofrontal and insular cortices (p < 0.05) correlated inversely with the duration of methamphetamine abstinence. These results suggest that chronic self-administration of methamphetamine can cause reactive microgliosis in the brains of human methamphetamine abusers, a level of activation that appears to subside over longer periods of abstinence.

In vivo changes in microglial activation and amyloid deposits in brain regions with hypometabolism in Alzheimer’s disease

PurposeAmyloid β protein (Aβ) is known as a pathological substance in Alzheimer’s disease (AD) and is assumed to coexist with a degree of activated microglia in the brain. However, it remains unclear whether these two events occur in parallel with characteristic hypometabolism in AD in vivo. The purpose of the present study was to clarify the in vivo relationship between Aβ accumulation and neuroinflammation in those specific brain regions in early AD.MethodsEleven nootropic drug-naïve AD patients underwent a series of positron emission tomography (PET) measurements with [11C](R)PK11195, [11C]PIB and [18F]FDG and a battery of cognitive tests within the same day. The binding potentials (BPs) of [11C](R)PK11195 were directly compared with those of [11C]PIB in the brain regions with reduced glucose metabolism.ResultsBPs of [11C](R)PK11195 and [11C]PIB were significantly higher in the parietotemporal regions of AD patients than in ten healthy controls. In AD patients, there was a negative correlation between dementia score and [11C](R)PK11195 BPs, but not [11C]PIB, in the limbic, precuneus and prefrontal regions. Direct comparisons showed a significant negative correlation between [11C](R)PK11195 and [11C]PIB BPs in the posterior cingulate cortex (PCC) (p < 0.05, corrected) that manifested the most severe reduction in [18F]FDG uptake.ConclusionA lack of coupling between microglial activation and amyloid deposits may indicate that Aβ accumulation shown by [11C]PIB is not always the primary cause of microglial activation, but rather the negative correlation present in the PCC suggests that microglia can show higher activation during the production of Aβ in early AD.

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model.

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the L-glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.

Altered Brain Serotonin Transporter and Associated Glucose Metabolism in Alzheimer Disease

Whether preclinical depression is one of the pathophysiologic features of Alzheimer disease (AD) has been under debate. In vivo molecular imaging helps clarify this kind of issue. Here, we examined in vivo changes in the brain serotoninergic system and glucose metabolism by scanning early- to moderate-stage AD patients with and without depression using PET with a radiotracer for the serotonin transporter, 11C-3-amino-4-(2-dimethylaminomethylphenylsulfanyl) benzonitrile (DASB), and a metabolic marker, 18F-FDG. Methods: Fifteen AD patients (8 nondepressed and 7 depressed) and 10 healthy subjects participated. All participants underwent 3-dimensional MRI and quantitative 11C-DASB PET measurements, followed by 18F-FDG PET scans in the AD group. Region-of-interest analysis was used to examine changes in 11C-DASB binding potential estimated quantitatively by the Logan plot method in the serotonergic projection region. In addition, statistical parametric mapping was used to examine whether glucose metabolism in any brain region correlated with levels of 11C-DASB binding in the dense serotonergic projection region (striatum) in AD. Results: Psychologic evaluation showed that general cognitive function (Mini-Mental State Examination) was similar between the 2 AD subgroups. Striatal 11C-DASB binding was significantly lower in AD patients, irrespective of depression, than in healthy controls (P < 0.05, corrected), and 11C-DASB binding in other dense projection areas decreased significantly in the depressive group, compared with the control group. The 11C-DASB binding potential levels in the subcortical serotonergic projection region correlated negatively with depression score (Spearman correlation, P < 0.01) but not with dementia score. Statistical parametric mapping correlation analysis showed that glucose metabolism in the right dorsolateral prefrontal cortex was positively associated with the level of striatal 11C-DASB binding in AD. Conclusion: The significant reduction in 11C-DASB binding in nondepressed AD patients suggests that presynaptic serotonergic function is altered before the development of psychiatric problems such as depression in AD. The depressive AD group showed greater and broader reductions in binding, suggesting that a greater loss of serotonergic function relates to more severe psychiatric symptoms in the disease. This serotonergic dysfunction may affect the activity of the right dorsolateral prefrontal cortex, a higher center of cognition and emotion in AD.

Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+ oscillations

Glial contribution to in vitro synaptic function was investigated in a neuron-glia co-culture system by monitoring spontaneous oscillations of intracellular Ca2+ in neurons. Rat cortical neurons, grown stably on a cortical astrocyte monolayer, extended neurites resulting in marked functional synapse formation. Little synapse formation was observed in neuronal co-culture with meningeal fibroblasts or endothelial cells. Aged astrocytes in vitro (C35) were found to attenuate synaptic development, while young astrocytes (C5) markedly promoted synaptic function. C5 and C35 astrocyte media conditioned yielded no significant synaptogenic effect, indicating diffusible factor(s) are not responsible for our observation. Modulation of astrocytic proliferation and differentiation by gliostatin, a glial growth inhibitor, or dibutyryl cAMP affected neuronal synaptic function on the co-cultures. Site-specific analysis in homologous and heterologous neuron-astrocyte co-cultures among cortex, hippocampus, septum, and striatum revealed that homologous combinations of neurons and astrocytes derived from identical brain regions elicited the largest number of synchronizing neurons. These results suggest that in vivo neuronal synaptic function essentially requires the participation of adjacent astrocytes, which is site-specific and age-dependent.

Differential expression of KCC2 accounts for the differential GABA responses between relay and intrinsic neurons in the early postnatal rat olfactory bulb.

The rat olfactory bulb is anatomically immature at birth, and considerable neurogenesis and synaptogenesis are known to take place postnatally. In addition, significant physiological changes have also been reported in this period. For example, granule cell‐mediated inhibition following electrical stimulations to the lateral olfactory tract is robust during the first postnatal week, and then decreases abruptly after the second week. However, the mechanism underlying this enhanced inhibition remains to be elucidated. To know the cause of this phenomenon, we investigated the expression patterns of cation‐Cl– co‐transporters (KCC1, KCC2 and NKCC1) mRNAs, which are responsible for the regulation of [Cl–]i. In addition, responses to γ‐aminobutyric acid (GABA) were measured by gramicidin‐perforated patch‐clamp recordings and Ca2+ imaging using fura‐2. We found that in the early postnatal period, mitral cells expressing KCC2 mRNA were inhibited by GABA, while granule cells lacking KCC2 mRNA expression were depolarized or excited by GABA. These results indicate that transient GABA‐mediated excitation on granule cells might be the main cause of the enhanced inhibition on mitral cells, and suggest that these differential GABA responses between relay and intrinsic neurons play pivotal roles in the early postnatal rat olfactory bulb.

Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP–CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

Glycine cleavage system in neurogenic regions.

The glycine cleavage system (GCS) is the essential enzyme complex for degrading glycine and supplying 5,10‐methylenetetrahydrofolate for DNA synthesis. Inherited deficiency of this system causes nonketotic hyperglycinemia, characterized by severe neurological symptoms and frequent association of brain malformations. Although high levels of glycine have been considered to cause the above‐mentioned problems, the detailed pathogenesis of this disease is still unknown. Here we show that GCS is abundantly expressed in rat embryonic neural stem/progenitor cells in the neuroepithelium, and this expression is transmitted to the radial glia–astrocyte lineage, with prominence in postnatal neurogenic regions. These data indicate that GCS plays important roles in neurogenesis, and suggest that disturbance of neurogenesis induced by deficiency of GCS may be the main pathogenesis of nonketotic hyperglycinemia.

Alterations in α4β2 nicotinic receptors in cognitive decline in Alzheimer’s aetiopathology

Nicotinic acetylcholine receptor subtype α4β2 is considered important in the regulation of attention and memory, and cholinergic degeneration is known as one pathophysiology of Alzheimers disease. Brain amyloid-β protein deposition is also a key pathological marker of Alzheimers disease. Recent amyloid-β imaging has shown many cognitively normal subjects with amyloid-β deposits, indicating a missing link between amyloid-β deposition and cognitive decline. To date, the relationship between the α4β2 nicotinic acetylcholine receptor and amyloid-β burden has not been elucidated in vivo. In this study we investigated the relation between α4β2 nicotinic acetylcholine receptor availability in the brain, cognitive functions and amyloid-β burden in 20 non-smoking patients with Alzheimers disease at an early stage and 25 age-matched non-smoking healthy elderly adults by measuring levels of α4β2 nicotinic acetylcholine receptor binding estimated from a simplified ratio method (BPRI) and Logan plot-based amyloid-β accumulation (BPND) using positron emission tomography with α4β2 nicotinic acetylcholine receptor tracer (18)F-2FA-85380 and (11)C-Pittsburgh compound B. The levels of tracer binding were compared with clinical measures for various brain functions (general cognition, episodic and spatial memory, execution, judgement, emotion) using regions of interest and statistical parametric mapping analyses. Between-group statistical parametric mapping analysis showed a significant reduction in (18)F-2FA-85380 BPRI in the cholinergic projection region in patients with Alzheimers disease with a variety of (11)C-Pittsburgh compound B accumulation. Spearman rank correlation analyses showed positive correlations of (18)F-2FA-85380 BPRI values in the medial frontal cortex and nucleus basalis magnocellularis region with scores of the Frontal Assessment Battery (a test battery for executive functions and judgement) in the Alzheimers disease group (P < 0.05 corrected for multiple comparison), and also positive correlations of the prefrontal and superior parietal (18)F-2FA-85380 BPRI values with the Frontal Assessment Battery score in the normal group (P < 0.05 corrected for multiple comparison). These positive correlations indicated an in vivo α4β2 nicotinic acetylcholine receptor role in those specific functions that may be different from memory. Both region of interest-based and voxelwise regression analyses showed a negative correlation between frontal (11)C-Pittsburgh compound B BPND and (18)F-2FA-85380 BPRI values in the medial frontal cortex and nucleus basalis magnocellularis region in patients with Alzheimers disease (P < 0.05 corrected for multiple comparison). These findings suggest that an impairment of the cholinergic α4β2 nicotinic acetylcholine receptor system with the greater amount of amyloid deposition in the system plays an important role in the pathophysiology of Alzheimers disease.

Elevated Transcription Factor Specificity Protein 1 in Autistic Brains Alters the Expression of Autism Candidate Genes

BACKGROUND Profound changes in gene expression can result from abnormalities in the concentrations of sequence-specific transcription factors like specificity protein 1 (Sp1). Specificity protein 1 binding sites have been reported in the promoter regions of several genes implicated in autism. We hypothesize that dysfunction of Sp1 could affect the expression of multiple autism candidate genes, contributing to the heterogeneity of autism. METHODS We assessed any alterations in the expression of Sp1 and that of autism candidate genes in the postmortem brain (anterior cingulate gyrus [ACG], motor cortex, and thalamus) of autism patients (n = 8) compared with healthy control subjects (n = 13). Alterations in the expression of candidate genes upon Sp1/DNA binding inhibition with mithramycin and Sp1 silencing by RNAi were studied in SK-N-SH neuronal cells. RESULTS We observed elevated expression of Sp1 in ACG of autism patients (p = .010). We also observed altered expression of several autism candidate genes. GABRB3, RELN, and HTR2A showed reduced expression, whereas CD38, ITGB3, MAOA, MECP2, OXTR, and PTEN showed elevated expression in autism. In SK-N-SH cells, OXTR, PTEN, and RELN showed reduced expression upon Sp1/DNA binding inhibition and Sp1 silencing. The RNA integrity number was not available for any of the samples. CONCLUSIONS Transcription factor Sp1 is dysfunctional in the ACG of autistic brain. Consequently, the expression of potential autism candidate genes regulated by Sp1, especially OXTR and PTEN, could be affected. The diverse downstream pathways mediated by the Sp1-regulated genes, along with the environmental and intracellular signal-related regulation of Sp1, could explain the complex phenotypes associated with autism.