The lysosomal enzyme alpha-Galactosidase A is deficient in Parkinson's disease brain in association with the pathologic accumulation of alpha-synuclein.

The aberrant accumulation of alpha-synuclein (α-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of α-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of α-syn. We previously identified the pathological accumulation of α-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (α-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified α-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to α-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in α-Gal A activity and the 46kDa "active" species of α-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in α-Gal A activity/levels correlated significantly with increased α-syn phosphorylated at serine 129 (p129S-α-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb3), a substrate of α-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb3 isoforms and with decreased α-Gal A activity and/or increased p129S-α-syn. Deacylated Gb3 (globotriaosylsphingosine or lyso-Gb3) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a significant correlation was observed between decreasing GCase activity and increasing p129S-α-syn. Together our findings indicate α-Gal A deficiency in late-stage PD brain that correlates significantly with the pathological accumulation of α-syn, and further suggest the potential for α-Gal A and its glycosphingolipid substrates as putative biomarkers for PD.

Impact of neonatal NOS-1 inhibitor exposure on neurobehavioural measures and prefrontal-temporolimbic integration in the rat nucleus accumbens.

Nitric oxide (NO) is a gaseous neurotransmitter that plays a significant role in the establishment and refinement of functional neural circuits. Genetic and post-mortem studies have suggested that neuronal NO synthase (NOS-1) activity may be compromised in frontal and temporal lobes, and related structures, in schizophrenia. The goal of this study was to determine if there is a link between neonatal disruptions in NO signalling and disturbances in the development and function of prefrontal-temporolimbic circuits. Neonatal rats were injected on postnatal days PD3-5 with the selective NOS-1 inhibitor Nω-propyl-L-arginine (NPA) and tested in adulthood (≥PD60) or as juveniles (PD30). Adult rats treated with NPA as neonates exhibited increased amphetamine-induced locomotion compared to animals receiving vehicle as neonates, whereas this was not observed in juvenile rats treated with NPA as neonates. Adult rats exposed to NPA as neonates also exhibited deficits in social interaction and short-term recognition memory, as well as reduced brain weight, compared to vehicle-treated controls. Finally, neonatal NPA exposure increased the responsiveness of nucleus accumbens neurons to prefrontal cortical input and disrupted the modulation of cortico-accumbens circuits by hippocampal afferents that is normally observed in adult animals. These results show for the first time that neonatal inhibition of NOS-1 during a critical neurodevelopmental period leads to aberrant behaviours that manifest in adulthood, as well as electrophysiological abnormalities in prefrontal-temporolimbic circuits. Greater understanding of the role of NOS-1 in the development of these circuits will shed light on how developmental insults translate to pathophysiology associated with schizophrenia.

Brain expression level and activity of HDAC6 protein in neurodegenerative dementia.

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic protein that plays an especially critical role in the formation of aggresomes, where aggregates of excess protein are deposited. Previous immunohistochemical studies have shown that HDAC6 accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies (DLB) as well as in glial cytoplasmic inclusions in multiple system atrophy (MSA). However, it is uncertain whether the level and activity of HDAC6 are altered in the brains of patients with neurodegenerative dementia. In the present study, we demonstrated that the level of HDAC6 was not altered in the temporal cortex of patients with Alzheimer's disease and DLB in comparison with controls. In contrast, the level of HDAC6 was significantly increased in the temporal cortex of patients with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in the cerebellar white matter of patients with MSA. However, the level of acetylated α-tubulin, one of the substrates of HDAC6, was not altered in FTLD-TDP and MSA relative to controls. These findings suggest that the induced level of HDAC6 in the brain is insufficient for manifestation of its activity in FTLD-TDP and MSA.

Aluminum and ethanol induce alterations in superoxide and peroxide handling capacity (SPHC) in frontal and temporal cortex.

Aluminum is an omnipresent neurotoxicant and has been associated with several neuropathological disorders. Cerebrum and cerebellum have been shown to face augmented oxidative stress when animals are exposed to aluminum and high doses of ethanol. To establish the link between oxidative stress and neurobehavioral alterations, the present study was conducted to determine the extent of oxidative stress in low levels of pro-oxidant (ethanol exposure) status of the functionally discrete regions of the cerebrum. Male Wistar rats were exposed to aluminum (10 mg/kg body wt) and ethanol (0.2-0.6 g/kg body wt) for 4 weeks. Spontaneous motor activity (SMA) and Rota-Rod performances (RRP) were recorded weekly during the period of exposure. At the end of 4th week, oxidative stress parameters were determined from the homogenized cerebral tissue. GSH-independent superoxide peroxide handling capacity (GI-SPHC) and GSH-dependent superoxide peroxide handling capacity (GD-SPHC) were determined for FC and TC upon exposure to ethanol in the absence and presence of aluminum exposure. Aluminum was found to augment the oxidative stress at higher doses (0.6 g Ethanol/kg body wt) of ethanol, particularly in FC. The SPHC of FC was also found to be compromised significantly in aluminum-ethanol co-exposed animals. It was concluded that even though the manifestation of oxidative stress was not observed as revealed by assaying the widely used oxidative stress biochemical markers (indices), aluminum and ethanol (low doses) exposure induced alterations in the handling capacity of oxidant imbalance that could be recognized by studying the SPHC of FC. Comparison of GD-SPHC and GI-SPHC offered a possible mechanism of compromised SPHC in FC. This observation is likely to offer insights into the mechanism of association between aluminium exposure and behavioral changes in neurodegenerative disorders towards therapeutic strategies for these disorders.

Differential BDNF signaling in dentate gyrus and perirhinal cortex during consolidation of recognition memory in the rat.

Consolidation of long-term memory is dependent on synthesis of new proteins in the hippocampus and associated cortical regions. The neurotrophin brain-derived neurotrophic factor (BDNF) is tightly regulated by activity-dependent cellular processes and is strongly linked with mechanisms underlying learning and memory. BDNF activation of tyrosine receptor kinase (TrkB) stimulates intracellular signaling cascades implicated in plasticity, including the extracellular-signal related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositide-3-kinase (PI3K)/Akt pathway. Here, we investigate the role of BDNF, ERK/MAPK, and PI3K/AKT signaling cascade in recognition memory in the rat. We report that recognition memory was associated with increased release of BDNF in the dentate gyrus and perirhinal cortex. This was associated with significant increases in p44ERK activation and c-fos expression in the dentate gyrus and PI3K activation and c-fos expression in the perirhinal cortex. Furthermore, both recognition memory and the associated cell signaling events in dentate gyrus and perirhinal cortex were blocked by intraperitoneal injection of the Trk receptor inhibitor tyrphostin AG879. These data are consistent with the hypothesis that BDNF-stimulated intracellular signaling plays a role in consolidation of recognition memory in the rat.

Increased expression of histone deacetylases 2 in temporal lobe epilepsy: a study of epileptic patients and rat models.

Histone deacetylases 2 (HDAC2) is expressed in the central nervous system; it has multiple functions in neural plasticity. However, we do not know if HDAC2 is also involved in the pathology of epilepsy. Here we report that HDAC2 was expressed in the brain tissues of both control and temporal lobe epilepsy (TLE) patients. Results from immunofluorescence and immunohistochemistry showed that HDAC2 was primarily located in the nucleus and that TLE patients exhibit significantly more HDAC2 positive cells than control. Western blotting showed that HDAC2 protein levels were significantly higher in TLE than in control brain. Moreover, in the rat model of TLE, there was a sustained enhancement of HDAC2 expression in rat models of TLE. HDAC2 was significantly increased in both the acute (1 day) and chronic (60 days) animals compared with control group. These results suggest that HDAC2 play an important role in the pathogenesis of human TLE.

Study of lipid profile and parieto-temporal lipid peroxidation in AlCl3 mediated neurotoxicity. Modulatory effect of fenugreek seeds.

BACKGROUND: Peroxidation of lipid (LPO) membrane and cholesterol metabolism have been involved in the physiopathology of many diseases of aging brain. Therefore, this prospective animal study was carried firstly to find out the correlation between LPO in posterior brain and plasmatic cholesterol along with lipoprotein levels after chronic intoxication by aluminium chloride (AlCl3). Chronic aluminum-induced neurotoxicity has been in fact related to enhanced brain lipid peroxidation together with hypercholesterolemia and hypertriglyceridemia, despite its controversial etiological role in neurodegenerative diseases. Secondly an evaluation of the effectiveness of fenugreek seeds in alleviating the engendered toxicity through these biochemical parameters was made. RESULTS: Oral administration of AlCl3 to rats during 5 months (500 mg/kg bw i.g for one month then 1600 ppm via the drinking water) enhanced the levels of LPO in posterior brain, liver and plasma together with lactate dehydrogenase (LDH) activities, total cholesterol (TC), triglycerides (TG) and LDL-C (Low Density Lipoproteins) levels. All these parameters were decreased following fenugreek seeds supplementation either as fenugreek seed powder (FSP) or fenugreek seed extract (FSE). A notable significant correlation was observed between LPObrain and LDL-C on one hand and LDHliver on the other hand. This latter was found to correlate positively with TC, TG and LDL-C. Furthermore, high significant correlations were observed between LDHbrain and TC, TG, LDL-C, LPObrain as well as LDHliver. CONCLUSION: Aluminium-induced LPO in brain could arise from alteration of lipid metabolism particularly altered lipoprotein metabolism rather than a direct effect of cholesterol oxidation. Fenugreek seeds could play an anti-peroxidative role in brain which may be attributed in part to its modulatory effect on plasmatic lipid metabolism.

High Expression of Nicotinamide N-Methyltransferase in Patients with Sporadic Alzheimer's Disease.

We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson's disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer's disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms.

Comprehensive Gene Expression Analysis Detects Global Reduction of Proteasome Subunits in Schizophrenia.

BACKGROUND: The main challenge in the study of schizophrenia is its high heterogeneity. While it is generally accepted that there exist several biological mechanisms that may define distinct schizophrenia subtypes, they have not been identified yet. We performed comprehensive gene expression analysis to search for molecular signals that differentiate schizophrenia patients from healthy controls and examined whether an identified signal was concentrated in a subgroup of the patients. METHODS: Transcriptome sequencing of 14 superior temporal gyrus (STG) samples of subjects with schizophrenia and 15 matched controls from the Stanley Medical Research Institute (SMRI) was performed. Differential expression and pathway enrichment analysis results were compared to an independent cohort. Replicability was tested on 6 additional independent datasets. RESULTS: The 2 STG cohorts showed high replicability. Pathway enrichment analysis of the down-regulated genes pointed to proteasome-related pathways. Meta-analysis of differential expression identified down-regulation of 12 of 39 proteasome subunit genes in schizophrenia. The signal of proteasome subunits down-regulation was replicated in 6 additional datasets (overall 8 cohorts with 267 schizophrenia and 266 control samples, from 5 brain regions). The signal was concentrated in a subgroup of patients with schizophrenia. CONCLUSIONS: We detected global down-regulation of proteasome subunits in a subgroup of patients with schizophrenia. We hypothesize that the down-regulation of proteasome subunits leads to proteasome dysfunction that causes accumulation of ubiquitinated proteins, which has been recently detected in a subgroup of schizophrenia patients. Thus, down-regulation of proteasome subunits might define a biological subtype of schizophrenia.

Differential proteomics analysis of specific carbonylated proteins in the temporal cortex of aged rats: the deterioration of antioxidant system.

Oxidative stress plays a pivotal role in normal brain aging and various neurodegenerative diseases, including Alzheimer's disease (AD). Irreversible protein carbonylation, a widely used marker for oxidative stress, rises during aging. The temporal cortex is essential for learning and memory and particularly susceptible to oxidative stress during aging and in AD patients. In this study, we used 2-DE, MALDI-TOF/TOF MS, and Western blotting to analyze the differentially carbonylated proteins in the rat temporal cortex between 1-month-old and 24-month-old. We showed that the carbonyl levels of ten protein spots corresponding to six gene products: SOD1, SOD2, peroxiredoxin 1, peptidylprolyl isomerase A, cofilin 1, and adenylate kinase 1, significantly increased in the temporal cortex of aged rats. These proteins are associated with antioxidant defense, the cytoskeleton, and energy metabolism. Several oxidized proteins identified in aged rat brain are known to be involved in neurodegenerative disorders as well. Our findings indicate that these carbonylated proteins may be implicated in the decline of normal brain aging process and provide insights into the mechanisms underlying age-associated dysfunction of temporal cortex.

Temporal lobe sclerosis associated with hippocampal sclerosis in temporal lobe epilepsy: neuropathological features.

Widespread changes involving neocortical and mesial temporal lobe structures can be present in patients with temporal lobe epilepsy and hippocampal sclerosis. The incidence, pathology, and clinical significance of neocortical temporal lobe sclerosis (TLS) are not well characterized. We identified TLS in 30 of 272 surgically treated cases of hippocampal sclerosis. Temporal lobe sclerosis was defined by variable reduction of neurons from cortical layers II/III and laminar gliosis; it was typically accompanied by additional architectural abnormalities of layer II, that is, abnormal neuronal orientation and aggregation. Quantitative analysis including tessellation methods for the distribution of layer II neurons supported these observations. In 40% of cases, there was a gradient of TLS with more severe involvement toward the temporal pole, possibly signifying involvement of hippocampal projection pathways. There was a history of a febrile seizure as an initial precipitating injury in 73% of patients with TLS compared with 36% without TLS; no other clinical differences between TLS and non-TLS cases were identified. Temporal lobe sclerosis was not evident preoperatively by neuroimaging. No obvious effect of TLS on seizure outcome was noted after temporal lobe resection; 73% became seizure-free at 2-year follow-up. In conclusion, approximately 11% of surgically treated hippocampal sclerosis is accompanied by TLS. Temporal lobe sclerosis is likely an acquired process with accompanying reorganizational dysplasia and an extension of mesial temporal sclerosis rather than a separate pathological entity.

Amyloid-beta(25-35) impairs memory and increases NO in the temporal cortex of rats.

beta-Amyloid plays an important role in the neurodegeneration process of Alzheimer's disease (AD), but its neurotoxic mechanisms are not clear. It has been associated with the increase of oxidative stress and cognitive impairment because the beta-amyloid peptide 25-35 (Abeta((25-35))) has the critical neurotoxic properties of the full-length Abeta(1-42). Our present study shows the role of Abeta((25-35)) when injected into the temporal cortex on the nitric oxide pathways, 3-nitrotyrosine, neuronal death, and the spatial memory of rats 1 month after the injection. Our data showed that Abeta((25-35)) increases oxidative stress, causes neuronal damage, and decreases spatial memory in rats. Notably, the injection of the fraction Abeta((25-35)) caused an increase of nNOS and iNOS immunoreactivity in the temporal cortex and hippocampus. We demonstrated a significant increase of reactive astrocytosis, which was accompanied by neuronal damage in the temporal cortex and hippocampus of rats injected with Abeta((25-35)). These data suggest that the fraction Abeta((25-35)) injected into the temporal cortex might contribute to understanding the role of nitric oxide on the biological changes related to the neuropathological progression and the memory impairment in AD.

Parkinson's disease: activity of L-dopa decarboxylase in discrete brain regions.

The activity of L-dopa decarboxylase was greatly reduced in the striatum, less so in the hypothalamus, and unchanged in the cortex of brains of patients with Parkinson's disease. However, it appears that even in the striatum enough activity remained to allow for the formation of dopamine from L-dopa in patients treated with large doses of L-dopa.

Choline acetyltransferase activity in vascular dementia and stroke.

BACKGROUND/AIM: Alterations in cholinergic activity have not been systematically studied in types of cerebrovascular disease. We examined cholinergic function at postmortem, focussing on stroke and vascular dementia (VaD). METHODS: Post-mortem brain tissue was studied from 61 patients with stroke or VaD (13 infarct dementia; 8 stroke/no dementia; 11 sub-cortical ischaemic VaD, SIVD; 29 VaD and concurrent Alzheimer's disease, AD), 12 patients with AD and 23 controls. Choline acetyltransferase (ChAT) was measured in Brodmann areas (BA) 9 and 20/21. RESULTS: There were significant reductions in ChAT activity in patients with VaD and concurrent AD compared to age-matched controls (BA9: t = 2.7, p = 0.009; BA20/21: t = 4.67, p = 0.000). In patients with infarct dementia, there was a significant 27% increase in ChAT activity in BA9 (t = 2.1, p = 0.047), but not in BA20/21 (t = 1.67, p = 0.106), compared to the age-matched control group. There was no relationship between ChAT activity and cognition in the VaD patients. CONCLUSIONS: Loss of cholinergic function is only evident in VaD patients with concurrent AD. A novel increase in cholinergic activity was identified in patients with infarct dementia, which may create important new treatment opportunities.

Na+, K+-ATPase α3 isoform in frontal cortex GABAergic neurons in psychiatric diseases.

Na+, K+-ATPase is an essential membrane transporter. In the brain, the α3 isoform of Na+, K+-ATPase is vital for neuronal function. The enzyme and its regulators, endogenous cardiac steroids (ECS), were implicated in neuropsychiatric disorders. GABAergic neurotransmission was also studied extensively in diseases such as schizophrenia and bipolar disorder (BD). Post mortem brain samples from subjects with depression, schizophrenia or BD and non-psychiatric controls were provided by the Stanley Medical Research Institute. ECS levels were determined by ELISA. Expression levels of the three Na+, K+-ATPase-α isoforms, α1, α2 and α3, were determined by Western blot analysis. The α3 levels in GABAergic neurons in different regions of the brain were quantified by fluorescence immunohistochemistry. The results show that Na+, K+ -ATPase α3 isoform levels were lower in GABAergic neurons in the frontal cortex in BD and schizophrenia as compared with the controls (n = 15 subjects per group). A study on a 'mini-cohort' (n = 3 subjects per group) showed that the α3 isoform levels were also lower in GABAergic neurons in the hippocampus, but not amygdala, of bipolar and schizophrenic subjects. In the temporal cortex, higher Na+, K+ -ATPase α3 protein levels were found in the three psychiatric groups. No significant differences in ECS levels were found in this brain area. This is the first report on the distribution of α3 in specific neurons in the human brain in association with mental illness. These results strengthen the hypothesis for the involvement of Na+, K+ -ATPase in neuropsychiatric diseases.

Upregulation of GSK3beta expression in frontal and temporal cortex in ALS with cognitive impairment (ALSci).

The deposition of highly phosphorylated microtubule-associated tau protein has been observed in ALS with cognitive impairment (ALSci). In these studies, we have examined whether the expression of two candidate protein kinases for mediating tau hyperphosphorylation (GSK3beta or CDK5) are also altered. The expression of GSK, CDK and p25/p35 was assayed in human frontal, hippocampal, cerebellar, cervical (dorsal and ventral) and lumbar (dorsal and ventral) tissue from neurologically intact control (5), ALS (5) or ALSci (5) patients using RT-PCR, Western blot or immunohistochemistry. To assess GSK-3beta activity, we examined GSK3beta, phospho-GSK3beta and phospho-beta-catenin expression. Expression levels relative to that of beta-actin were compared by ANOVA. The expression of GSK, GSK3beta and phospho-GSK3beta was increased in both ALS and ALSci compared to that of the control. This was accompanied by an increased expression of phospho-beta-catenin. No significant difference between control, ALS or ALSci was observed with respect to the expression of CDK5 or p25/p35. Both GSK3beta and phospho-GSK3beta immunoreactive neurons were mainly located in layer II and layer III in the frontal cortex and in layer II in the hippocampus. This was consistent with the previously described distribution of hyperphosphorylated tau bearing neurons in ALS and ALSci. These data suggest that GSK3beta expression is upregulated in ALS and ALSci and that GSK3beta activation is associated with the intraneuronal deposition of hyperphosphorylated tau protein. This supports the potential role for GSK3beta as a therapeutic target in ALS.

Aromatase distribution in the monkey temporal neocortex and hippocampus.

Numerous studies have shown that neuronal plasticity in the hippocampus and neocortex is regulated by estrogen and that aromatase, the key enzyme for estrogen biosynthesis, is present in cerebral cortex. Although the expression pattern of aromatase mRNA has been described in the monkey brain, its precise cellular distribution has not been determined. In addition, the degree to which neuronal aromatase is affected by gonadal estrogen has not been investigated. In this study, we examined the immunohistochemical distribution of aromatase in young ovariectomized female rhesus monkeys with or without long-term cyclic estradiol treatment. Both experimental groups showed that aromatase is localized in a large population of CA1-3 pyramidal cells, in granule cells of the dentate gyrus and in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Moreover, numerous pyramidal cells were immunoreactive for aromatase in the neocortex, whereas only small subpopulations of neocortical interneurons were immunoreactive for aromatase. The widespread expression of the protein in a large neuronal population suggests that local intraneuroral estrogen synthesis may contribute to estrogen-induced synaptic plasticity in monkey hippocampus and neocortex of female rhesus monkeys. In addition, the apparent absence of obvious differences in aromatase distribution between the two experimental groups suggests that these localization patterns are not dependent on plasma estradiol levels.

Butyrylcholinesterase K variant associated with higher enzyme activity in the temporal cortex of elderly patients.

There is evidence to suggest an involvement of the K variant of the butyrylcholinesterase gene (BCHE) in dementia. We have examined the relationship between BCHE genotype and butyrylcholinesterase (BuChE) activity in autopsy brain tissue. We studied 164 autopsy cases, 144 with dementia and 20 controls, including 13 K homozygotes and 48 K heterozygotes, from three centres: Newcastle, Oxford and London. Mean BuChE activity in temporal cortex was 37% higher in K homozygotes than in wild-type homozygotes. Linear regression analysis, controlling for gender, diagnosis, age at death and study centre, showed that the number of BCHE-K alleles was associated with increasing BuChE activity (p=0.009).

Binding of 5'-O-(2-thiodiphosphate) to rat brain membranes is prevented by diadenosine tetraphosphate and correlates with ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) activity.

The distribution of binding sites for [(35)S]5'-O-(2-thiodiphosphate) ([(35)S]ADPbetaS), a radioligand of P2Y(1,12,13) receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K(d) values were in the range 1-2 nM. Diadenosine tetraphosphate (Ap(4)A) and diethenoadenosine tetraphosphate, epsilon-(Ap(4)A), displaced the radioligand, indicating dinucleotide binding to ADPbetaS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap(4)A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap(4)A signalling mediated by P2Y(1,12,13) receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain.

In vivo visualization of donepezil binding in the brain of patients with Alzheimer's disease.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * Deficit in central cholinergic neurotransmission is a consistent change associated with Alzheimer's disease (AD). * Donepezil hydrochloride exhibits selective inhibition of acetylcholinesterase (AChE) and is widely used for the treatment of AD. * The biodistribution of donepezil in the brain after administration is not precisely understood in vivo. * There is no method to measure the amount of binding of orally administered donepezil to AChE. WHAT THIS STUDY ADDS: * This study clearly visualizes the distribution of donepezil in human brain using [(11)C]-donepezil and positron emission tomography. * This study demonstrates prominent reduction of the donepezil binding site in the AD brain. * This study provides methodology to measure the AChE binding occupancy of orally administered donepezil and provides a new surrogate marker for evaluation and prediction of response to donepezil treatment. AIMS: The aims of this study were to visualize in vivo binding of donepezil to acetylcholinesterase (AChE) in the brain and to establish a method for measuring the amount of binding of orally administered donepezil. METHODS: [5-(11)C-methoxy]-donepezil ([(11)C]-donepezil) was radiolabelled as a positron emission tomography (PET) tracer. The biodistribution of [(11)C]-donepezil was measured by PET in 10 AD patients and six elderly normal subjects. Two AD patients underwent additional PET measurements after oral administration of donepezil for 6 months. RESULTS: [(11)C]-donepezil-PET images demonstrated high densities of tracer distribution in AChE-rich brain regions such as the striatum, thalamus, and cerebellum. Compared with elderly normal subjects, patients with mild AD exhibited about 18-20% reduction of donepezil binding in the neocortex and hippocampus, while patients with moderate AD exhibited about 24-30% reduction of donepezil binding throughout the brain. Orally administered donepezil (5 mg day(-1)) induced 61.6-63.3% reduction of donepezil binding in AD brains. The distribution volume of [(11)C]-donepezil in the hippocampus was significantly correlated with MMSE scores in AD patients. CONCLUSIONS: [(11)C]-donepezil-PET enables quantitative measurement of donepezil binding in the brain. AD patients exhibited reduction of donepezil binding in the brain, even in the early stage of disease. Longitudinal evaluation by this technique enables determination of AChE binding occupancy of orally administered donepezil.