Distinct binding of PET ligands PBB3 and AV-1451 to tau fibril strains in neurodegenerative tauopathies.

Diverse neurodegenerative disorders are characterized by deposition of tau fibrils composed of conformers (i.e. strains) unique to each illness. The development of tau imaging agents has enabled visualization of tau lesions in tauopathy patients, but the modes of their binding to different tau strains remain elusive. Here we compared binding of tau positron emission tomography ligands, PBB3 and AV-1451, by fluorescence, autoradiography and homogenate binding assays with homologous and heterologous blockades using tauopathy brain samples. Fluorescence microscopy demonstrated intense labelling of non-ghost and ghost tangles with PBB3 and AV-1451, while dystrophic neurites were more clearly detected by PBB3 in brains of Alzheimer's disease and diffuse neurofibrillary tangles with calcification, characterized by accumulation of all six tau isoforms. Correspondingly, partially distinct distributions of autoradiographic labelling of Alzheimer's disease slices with 11C-PBB3 and 18F-AV-1451 were noted. Neuronal and glial tau lesions comprised of 4-repeat isoforms in brains of progressive supranuclear palsy, corticobasal degeneration and familial tauopathy due to N279K tau mutation and 3-repeat isoforms in brains of Pick's disease and familial tauopathy due to G272V tau mutation were sensitively detected by PBB3 fluorescence in contrast to very weak AV-1451 signals. This was in line with moderate 11C-PBB3 versus faint 18F-AV-1451 autoradiographic labelling of these tissues. Radioligand binding to brain homogenates revealed multiple binding components with differential affinities for 11C-PBB3 and 18F-AV-1451, and higher availability of binding sites on progressive supranuclear palsy tau deposits for 11C-PBB3 than 18F-AV-1451. Our data indicate distinct selectivity of PBB3 compared to AV-1451 for diverse tau fibril strains. This highlights the more robust ability of PBB3 to capture wide-range tau pathologies.

Does Anticholinergic Activity Affect Neuropathology? Implication of Neuroinflammation in Alzheimer's Disease.

One characteristic neuropathological feature of Alzheimer's disease (AD) is profound neuronal loss in the nucleus basalis of Meynert, the major source of cholinergic innervation of the cerebral cortex. Clinically, anticholinergic activity causes a decline in cognitive function and increases the risk of dementia, thus possibly enhancing AD pathologies and neurodegeneration. Until now there has been insufficient human neuropathological data to support this conclusion. Experimental studies using a tauopathy mouse model demonstrated anticholinergics enhanced tau pathology and neurodegeneration corresponding to central anticholinergic activity. Additionally, donepezil, a cholinesterase inhibitor, ameliorated tau pathology and neurodegeneration in the same mouse model. These results indicate the balance between cholinergic and anticholinergic activities might affect neurodegeneration. Importantly, neurodegeneration observed in the mouse model seemed to correspond to the distribution of microglial activation, and it was reported that neuroinflammation plays an important role in the pathomechanism of AD, while anticholinergic activity augments inflammatory responses. Moreover, some studies indicated ß-amyloid itself depletes cholinergic function similarly to anticholinergic activity. Thus, anticholinergic activity might initiate and/or accelerate AD pathology. Limited human data support the conclusion that anticholinergic activity enhances AD-related neuropathology and neurodegeneration. However, experimental data from a tauopathy mouse model indicated anticholinergic activity might enhance neurodegeneration with enhanced neuroinflammation including microglial activation.

[Cognitive Function and Calcium. Suppression of neurotoxicity by an anti-dementia drug managing calcium influx].

Although drugs affecting N-methyl-D-aspartate (NMDA) receptors are being largely expected for anti-dementia drugs, memantine has been solely approved. As memantine has moderate affinity to NMDA receptors, memantine display efficacy of neuro-protection and learning promotion. In clinical trials, memantine showed suppression of progression of symptoms with dementia including improvement and suppression of aggressiveness and behavioral disturbance. Moreover, memantine can be prescribed with choline esterase inhibitors, thus, memantine is used as one of major anti-dementia drugs.

Effects of diet-induced obesity and voluntary exercise in a tauopathy mouse model: implications of persistent hyperleptinemia and enhanced astrocytic leptin receptor expression.

The number of patients with Alzheimer's disease (AD) is increasing worldwide, and available drugs have shown limited efficacy. Hence, preventive interventions and treatments for presymptomatic AD are currently considered very important. Obesity rates have also been increasing dramatically and it is an independent risk factor of AD. Therefore, for the prevention of AD, it is important to elucidate the pathomechanism between obesity and AD. We generated high calorie diet (HCD)-induced obese tauopathy model mice (PS19), which showed hyperleptinemia but limited insulin resistance. HCD enhanced tau pathology and glial activation. Conversely, voluntary exercise with a running wheel normalized the serum leptin concentration without reducing body weight, and restored the pathological changes induced by HCD. Thus, we speculated that persistent hyperleptinemia played an important role in accelerating pathological changes in PS19 mice. Leptin primarily regulates food intake and body weight via leptin receptor b (LepRb). Interestingly, the nuclear staining for p-STAT3, which was activated by LepRb, was decreased in hippocampal neurons in HCD PS19 mice, indicating leptin resistance. Meanwhile, astroglial activation and the astrocytic expression of a short LepR isoform, LepRa, were enhanced in the hippocampus of HCD PS19 mice. Real-time PCR analysis demonstrated that leptin increased mRNA levels for pro-inflammatory cytokines including IL-1ß and TNF-α in primary cultured astrocytes from wild type and LepRb-deficient mice. These observations suggest that persistent hyperleptinemia caused by obesity induces astrocytic activation, astrocytic leptin hypersensitivity with enhanced LepRa expression, and enhanced inflammation, consequently accelerating tau pathology in PS19 mice.

Therapeutic strategies for tau mediated neurodegeneration.

Based on the amyloid hypothesis, controlling ß-amyloid protein (Aß) accumulation is supposed to suppress downstream pathological events, tau accumulation, neurodegeneration and cognitive decline. However, in recent clinical trials, Aß removal or reducing Aß production has shown limited efficacy. Moreover, while active immunisation with Aß resulted in the clearance of Aß, it did not prevent tau pathology or neurodegeneration. This prompts the concern that it might be too late to employ Aß targeting therapies once tau mediated neurodegeneration has occurred. Therefore, it is timely and very important to develop tau directed therapies. The pathomechanisms of tau mediated neurodegeneration are unclear but hyperphosphorylation, oligomerisation, fibrillisation and propagation of tau pathology have been proposed as the likely pathological processes that induce loss of function or gain of toxic function of tau, causing neurodegeneration. Here we review the strategies for tau directed treatments based on recent progress in research on tau and our understanding of the pathomechanisms of tau mediated neurodegeneration.

Anticholinergics boost the pathological process of neurodegeneration with increased inflammation in a tauopathy mouse model.

Anticholinergics, and drugs with anticholinergic properties, are widely and frequently prescribed, especially to the elderly. It is well known that these drugs decrease cognitive function and increase the risk of dementia. Although the mechanism of anticholinergic drug-induced cognitive impairment has been assumed to be functionally reduced acetylcholine (ACh) neurotransmission, some data have indicated that anticholinergics might enhance the pathology of Alzheimer's disease. In this study, we investigated the pathological effects of anticholinergics on neurodegeneration. We chronically administered two anticholinergics, trihexyphenidyl (TP) and propiverine (PP) (the latter with less central anticholinergic action), to neurodegenerative tauopathy model mice 2 to 10 months old. Furthermore, because the ACh nervous system regulates both central and peripheral inflammation, we administered TP or PP to PS19 mice in which we had artificially induced inflammation by lipopolysaccharide injection. Tau pathology, synaptic loss, and neurodegeneration in the hippocampal region, as well as tau insolubility and phosphorylation, were markedly increased in TP-treated mice and mildly increased in PP-treated mice. Furthermore, immunohistochemical analysis revealed microglial proliferation and activation. Moreover, anticholinergics increased interleukin-1ß expression in both the spleen and brain of the tauopathy model mice intraperitoneally injected with lipopolysaccharide to induce systemic inflammation. Interestingly, these alterations were more strongly observed in TP-treated mice than in PP-treated mice, consistent with the level of central anticholinergic action. Anticholinergic drugs not only impair cognitive function by decreased ACh neurotransmission, but also accelerate neurodegeneration by suppressing an ACh-dependent anti-inflammatory system. Anticholinergics should be less readily prescribed to reduce the risk of dementia.

Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.

Filamentous tau inclusions are hallmarks of Alzheimer's disease (AD) and related tauopathies, but earlier pathologies may herald disease onset. To investigate this, we studied wild-type and P301S mutant human tau transgenic (Tg) mice. Filamentous tau lesions developed in P301S Tg mice at 6 months of age, and progressively accumulated in association with striking neuron loss as well as hippocampal and entorhinal cortical atrophy by 9-12 months of age. Remarkably, hippocampal synapse loss and impaired synaptic function were detected in 3 month old P301S Tg mice before fibrillary tau tangles emerged. Prominent microglial activation also preceded tangle formation. Importantly, immunosuppression of young P301S Tg mice with FK506 attenuated tau pathology and increased lifespan, thereby linking neuroinflammation to early progression of tauopathies. Thus, hippocampal synaptic pathology and microgliosis may be the earliest manifestations of neurodegenerative tauopathies, and abrogation of tau-induced microglial activation could retard progression of these disorders.

Analyses of natural courses of Japanese patients with Alzheimer's disease using placebo data from placebo-controlled, randomized clinical trials: Japanese Study on the Estimation of Clinical course of Alzheimer's disease.

INTRODUCTION: Symptomatic anti-Alzheimer's disease (AD) drugs have been commonly used for the treatment of AD. Knowing the natural courses of patients with AD on placebo is highly relevant for clinicians to understand their efficacy and for investigators to design clinical studies. METHODS: The data on rating scales for dementia such as Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) and Severe Impairment Battery were extracted from eight previous Japanese Phase II and III studies. Natural courses of Japanese AD patients in placebo groups were evaluated and statistically analyzed in a pooled and retrospective fashion. RESULTS: Decreases in ADAS-cog and Severe Impairment Battery was larger at week 22 or 24 than at week 12. Scores of ADAS-cog appeared to deteriorate faster in moderate AD than in mild AD. DISCUSSION: The present data will provide clinicians following up patients with AD with helpful information on how to manage AD patients and investigators with instruction for clinical study design.

Neuromyelitis optica and anti-aquaporin-4 antibodies measured by an enzyme-linked immunosorbent assay.

NMO-IgG, a disease-specific autoantibody for neuromyelitis optica, recognizes aquaporin-4 (AQP4) and has been examined by indirect immunofluorescence assay. We developed an enzyme-linked immunosorbent assay (ELISA) to detect anti-AQP4 antibodies by establishing methods for expression in a baculovirus system and purification of recombinant AQP4 as antigen. Elevated anti-AQP4 antibody titers in serum were found in 15 (71%) of 21 patients with neuromyelitis optica, 4.3% of 46 patients with multiple sclerosis, none of 51 normal controls, and 2.6% of 115 patients with other neurological diseases. The ELISA system can be substituted for the conventional NMO-IgG assay.

[Neurodegeneration and inflammation: analysis of a FTDP-17 model mouse].

Filamentous tau inclusions are hallmarks of Alzheimer's disease (AD) and related tauopathies, and the discovery of mutations in the tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) constitutes convincing evidence that tau proteins play a key role in the pathogenesis of neurodegenerative disorders. To investigate the pathomechanism of tauopathies, we generated and studied P301S mutant human tau transgenic mice (line PS19). Filamentous tau lesions developed in PS19 mice at 6-months of age, and progressively accumulated in association with striking neuron loss as well as hippocampal and entorhinal cortical atrophy by 9-12 months of age. Remarkably, hippocampal synapse loss and impaired synaptic function were detected in 3 month old PS19 mice before fibrillary tau tangles emerged. Prominent microglial activation and proinflammatory cytokine expressions in neurons also preceded tangle formation. Importantly, immunosuppression of young PS19 mice with FK506 attenuated tau pathology, thereby linking neuroinflammation to early progression of tauopathies. Recently, an anti-inflammatory function of acetylcholine (ACh) has been reported, suggesting that synaptic dysfunction might accelerate neuroinflammatory reaction by depletion of ACH. To investigate this, we administered donepezil (DZ), an ACh-esterase inhibitor, and trihexiphenidyl (TP), an anti-cholinergic agent to PS19 mice. Interestingly, DZ ameliorated but TP deteriorated microglial activation, tau pathology and neuronal loss, indicating the ACh level in the brain might play roles in not only neurotransmission, but also suppressing neuroinflammation in the brain.

Axonal degeneration induced by targeted expression of mutant human tau in oligodendrocytes of transgenic mice that model glial tauopathies.

Abundant filamentous tau inclusions in oligodendrocytes (OLGs) are hallmarks of neurodegenerative tauopathies, including sporadic corticobasal degeneration and hereditary frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). However, mechanisms of neurodegeneration in these tauopathies are unclear in part because of the lack of animal models for experimental analysis. We address this by generating transgenic (Tg) mice expressing human tau exclusively in OLGs using the 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter. Filamentous OLG tau inclusions developed in these Tg mice as a result of human tau expression in OLGs, especially those expressing the FTDP-17 human P301L mutant tau. Notably, structural disruption of myelin and axons preceded the emergence of thioflavin-S positive tau inclusions in OLGs, but impairments in axonal transport occurred even earlier, whereas motor deficits developed subsequently, especially in Tg mice with the highest tau expression levels. These data suggest that the accumulation of tau in OLG cause neurodegeneration, and we infer they do so by disrupting axonal transport. We suggest that similar defects may also occur in sporadic and hereditary human tauopathies with OLG tau pathologies.

HLA-DRB1*14 and DQB1*05 are associated with Japanese anti-MuSK antibody-positive myasthenia gravis patients.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disorder presumed to be associated with genetic susceptibility. This study aims to determine whether HLA is associated with MG in Japanese patients. METHODS: We included 58 MG patients with anti-acetylcholine receptor antibody (AChR+MG) and 14 MG patients with muscle-specific receptor tyrosine kinase (MuSK+MG) and determined HLA-A, B, DRB1 and -DQB1 alleles using polymerase chain reaction with sequence-specific oligonucleotide and primers. AChR+MG was classified into the three subgroups: early-onset MG (EOMG; n=11), late-onset MG (LOMG; n=20), and thymoma-associated MG (n=27). Healthy volunteers (n=100) served as controls. RESULTS: A significant positive association was observed between MuSK+MG with the DRB1*14 [57.1%, MuSK+MG vs. 18.0%, healthy controls (HC); odds ratio (OR): 6.1] and DQB1*05 [78.6%, MuSK+MG vs. 30.0%, HC; odds ratio (OR): 8.5]. We found a negative association between LOMG and DQB1*04 [5.0%, LOMG vs. 37.0%, HC; OR: 0.09]. There was no association between other MG subgroups and HLA alleles. CONCLUSION: HLA-DRB1*14 and DQB1*05 were associated with MuSK+MG, therefore these alleles may play important roles in developing MuSK+MG across the races.

Reduction of detyrosinated microtubules and Golgi fragmentation are linked to tau-induced degeneration in astrocytes.

Several human neurodegenerative diseases are associated with abnormal accumulations of aggregated tau proteins and glial degeneration in astrocytes, but the mechanism whereby tau proteins cause astrocytic degeneration is unclear. Here, we analyzed the biological consequences of overexpressing the longest human tau isoform in primary cultures of rat astrocytes using adenoviral-mediated gene transfer. Significantly, we found specific decreases in stable detyrosinated [glutamate (Glu)] microtubules (MTs) with concomitant increases in tubulin biosynthesis and the accumulation of acetylated, tyrosinated, alpha- and beta-tubulin. The consequences of this selective reduction in stable Glu-MTs included contemporaneous decreases in kinesin levels, collapse of the intermediate filament network, progressive disruption of kinesin-dependent trafficking of organelles, fragmentation of the Golgi apparatus that culminated in atrophy, and non-apoptotic death of astrocytes. These results suggest that reduced stable Glu-MTs is a primary consequence of tau accumulation that initiates mechanisms underlying astrocyte dysfunction and death in human neurodegenerative glial tauopathies.

Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy.

Intracellular accumulations of filamentous tau inclusions are neuropathological hallmarks of neurodegenerative diseases known as tauopathies. The discovery of multiple pathogenic tau gene mutations in many kindreds with familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) unequivocally confirmed the central role of tau abnormalities in the etiology of neurodegenerative disorders. To examine the effects of tau gene mutations and the role of tau abnormalities in neurodegenerative tauopathies, transgenic (Tg) mice were engineered to express the longest human tau isoform (T40) with or without the R406W mutation (RW and hWT Tg mice, respectively) that is pathogenic for FTDP-17 in several kindreds. RW but not hWT tau Tg mice developed an age-dependent accumulation of insoluble filamentous tau aggregates in neuronal perikarya of the cerebral cortex, hippocampus, cerebellum, and spinal cord. Significantly, CNS axons in RW mice contained reduced levels of tau when compared with hWT mice, and this was linked to retarded axonal transport and increased accumulation of an insoluble pool of RW but not hWT tau. Furthermore, RW but not hWT mice demonstrated neurodegeneration and a reduced lifespan. These data indicate that the R406W mutation causes reduced binding of this mutant tau to microtubules, resulting in slower axonal transport. This altered tau function caused by the RW mutation leads to increased accumulation and reduced solubility of RW tau in an age-dependent manner, culminating in the formation of filamentous intraneuronal tau aggregates similar to that observed in tauopathy patients.

Enhanced neurofibrillary tangle formation, cerebral atrophy, and cognitive deficits induced by repetitive mild brain injury in a transgenic tauopathy mouse model.

Traumatic brain injury (TBI) is a risk factors for Alzheimer's disease (AD), and repetitive TBI (rTBI) may culminate in dementia pugilistica (DP), a syndrome characterized by progressive dementia, parkinsonism, and the hallmark brain lesions of AD, including neurofibrillary tangles (NFTs), formed by abnormal tau filaments and senile plaques (SPs) composed of Abeta fibrils. Previous study showed that mild rTBI (mrTBI) accelerated the deposition of Abeta in the brains of transgenic (Tg) mice (Tg2576) that over-express human Abeta precursor proteins with the familial AD Swedish mutations (APP695swe) and model of AD-like amyloidosis. Here, we report studies of the effects of mrTBI on AD-like tau pathologies in Tg mice expressing the shortest human tau isoform (T44) subjected to mrTBI, causing brain concussion without structural brain damage to simulate injuries linked to DP. Twelve-month-old Tg T44 (n = 18) and wild-type (WT; n = 24) mice were subjected to mrTBI (four times a day, 1 day per week, for 4 weeks; n = 24) or sham treatment (n = 18). Histopathological analysis of mice at 9 months after mrTBI revealed that one of the Tg T44 mice showed extensive telencephalic NFT and cerebral atrophy. Although statistical analysis of neurobehavioral tests at 6 months after mrTBI did not show any significant difference in any of groups of mice, the Tg T44 mouse with extensive NFT had an exceptionally low neurobehavioral score. The reasons for the augmentation of tau pathologies in only one T44 tau Tg mouse subjected to mrTBI remain to be elucidated.

Expression of nitric oxide synthases in the anterior horn cells of amyotrophic lateral sclerosis.

The authors investigated for a correlation between the expression of nitric oxide synthases (NOSs) with the severity of motor neuronal loss in the anterior horns of patients with amyotrophic lateral sclerosis (ALS). Spinal cords from six patients with ALS and from three normal controls were examined. The sections of cervical, lumbar, and sacral cord including Onuf's nucleus, which are seldom degenerated until the late stage, were stained with three antibodies against NOSs (anti-n-NOS, anti-e-NOS, and anti-i-NOS) using ABC methods. Perikarya of motor neurons in ALS, but not in controls, were immunoreactive against anti-n-NOS and e-NOS. Anti-i-NOS did not recognize the motor neurons of ALS or of controls. The immunoreactivity for n- and e-NOSs was approximately the same in the sections of cervical, lumbar, and sacral cord in ALS. No significant differences in immunoreactivity were observed among the patients with ALS. These results suggest that the expression of NOSs does not immediately affect neuronal loss in ALS.

Low-dose tacrolimus treatment in thymectomised and steroid-dependent myasthenia gravis.

OBJECTIVES: The effects of tacrolimus, a new immunosuppressive drug, which inhibits calcineurin pathway and also might enhance corticosteroid (CS) receptor-mediated gene expressions, on clinical outcome and biochemical data were evaluated in thymectomised and steroid-dependent myasthenia gravis (MG) patients. PATIENTS AND METHODS: We administrated low-dose tacrolimus (3 mg/day orally) to 17 steroid-dependent thymectomised MG patients. They were followed for 4 to 58 months, average 19.2 months. The MG activities of daily living (MGADL) scores and the dosage of prednisolone (PSL) were assessed at baseline and 4 months later. RESULTS: The average MGADL scores improved from 6.8 to 5.0 (p < 0.01) at 4 months; to 3.5 at the last visit (p < 0.01) as well as the average dosage of PSL reducing from 31.6 to 24.1 mg/alternate day (p < 0.01) at 4 months; 14.6 mg at the last visit (p < 0.01). CONCLUSIONS: The additional low dose tacrolimus therapy for steroid-dependent thymectomised MG is effective in improving symptoms as well as allowing the tapering of CSs.

Increased ability of peripheral blood lymphocytes to degrade laminin in multiple sclerosis.

T lymphocytes and macrophages probably play a role in the pathogenesis of multiple sclerosis (MS), and migration of these cells into the central nervous system is facilitated by disruption of the capillary basement membrane. Laminin is a major extracellular matrix of the basement membrane. To investigate whether ability of lymphocytes to degrade laminin correlates with disease activity in MS, we conducted a prospective study in consecutive 24 MS patients. A novel quantitative assay was developed to estimate the ability of peripheral blood mononuclear cells (PBMCs) to degrade laminin. The assay was performed every four weeks over a period of 12 months. During the study period, a total of 41 relapses were observed. The ability to degrade laminin was significantly higher in MS patients, even during clinical remission, than in normal and neurological controls, and was transiently increased further within 4 weeks before relapse (p=0.076). In MS, the ability of peripheral blood lymphocytes to degrade laminin increases, and may correlate with disease activity.

Immunohistochemical analysis of tau phosphorylation and astroglial activation with enhanced leptin receptor expression in diet-induced obesity mouse hippocampus.

Accumulating evidence indicates that obesity is an independent risk factor for developing Alzheimer disease (AD). Recent studies have shown that diet-induced obesity (DIO) enhances AD-related pathologies in transgenic mouse models of the disease. DIO increases amyloid ß (Aß) deposition in amyloidogenic transgenic mice and enhances tau phosphorylation in tau transgenic mice. However, it remains unclear whether DIO also enhances AD-related pathological processes in wild-type (WT) mice. In this study, we examined the effects of DIO on Aß and tau pathology in WT mice using immunohistochemistry. In addition, we evaluated the protective effect of voluntary exercise on the DIO-induced pathological changes. DIO caused tau phosphorylation and astroglial activation in the hippocampus in WT mice. Interestingly, these changes were associated with enhanced astrocytic leptin receptor (LepR) expression and mild microgliosis, but not Aß accumulation. Although phosphorylated tau staining was only observed in the hippocampus, astrogliosis and microgliosis were present in both the amygdala and hippocampus. However, no apparent neuronal loss was observed. Voluntary exercise prevented these DIO-induced pathological changes. Our results demonstrate for the first time that DIO causes tau phosphorylation and that astrocytic LepR might be involved in the pathological process in WT mouse hippocampus. Our findings also suggest that physical exercise is a promising strategy for the prevention of AD in patients with obesity.