C型Niemann-Pick病神经原纤维缠结的形成与特征

目的 探讨C型尼曼-皮克病(NPC)脑部神经原纤维缠结(NFT)形成的时相特征.方法 以17例年龄7个月至55岁的NPC患者为研究对象,采用tau蛋白和有丝分裂期相关抗体进行免疫组织化学染色和银染,分析患者脑内NFT形成的特点.结果 最早可在4岁的患者海马旁回发现典型的NFT形成,随年龄增长数量逐渐增多.在形态上与阿尔茨海默病(AD)所见高度相似,但未发现老年斑.在NPC中,有丝分裂期磷酸化表位早于tau蛋白过度磷酸化及NFT形成.结论NFT形成并非老龄化过程的结果 ,且与老年斑的存在与否并无关联.cdc2/cyclinB1可能是NFT形成的关键性早期事件,针对其活性的抑制剂对于早期干预NPC NFT的形成有重要意义.     

Analysis of mouse model exhibiting neurofibrillary changes]

Dysfunction and filamentous microtubule-binding tau protein are key markers of neurodegenerative pathologies, including the pathology and neural degeneration associated with Alzheimer's disease (AD). Immunocytochemical studies of NFT-bearing neurons showed that NFTs are composed of ubiquitin and phosphorylation-dependent tau. Congo-red birefringency and thioflavin-S reactivity in NFT-bearing neurons also demonstrated that the tau aggregation forms a beta-sheet structure. Discovery of the molecular mechanisms of NFT formation may lead to more insight about events occurring during neurodegeneration. In frontotemporal dementia parkinsonism 17 (FTDP17), genetic studies indicated that tau is a causative gene, and mutation is found in exons and introns of tau gene. A patient who possesses this mutation exhibits pathologically NFT and clinically personality change and cognitive dysfunction. Then, we produced the Tg mice expressing human longest tau with missense mutation V337M. In the present study, neurons of hippocampus and cerebral cortex in our Tg mice showed phosphorylated and ubiquitinated tau aggregations with a beta-sheet structure. This was demonstrated by Congo-red and thioflavin-S positive staining, a histological criterion used to identify NFTs observed in neurodegenerative disorders. The mice also displayed altered behaviors that were associated with NFT formation. Thus, V337M mice provide a first animal model exhibiting altered behavior due to NFTs.     

Neurofibrillary tangles in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is an autosomal recessive disease, belonging to a clinically heterogeneous group of lipid storage diseases, distinguished by a unique error in cellular trafficking of exogenous cholesterol, associated with lysosomal accumulation of unesterified cholesterol. Unlike Niemann-Pick disease types A and B, there is no primary genetic defect in sphingomyelinase in NPC. During the routine neuropathological study of NPC patients, we found neurofibrillary tangles (NFT) in a series of cases with a slowly progressive chronic course. These were not associated with -amyloid deposits. The NFT were most frequent in the orbital gyrus, cingulate gyrus and entorhinal region of the cerebral cortex, but were also frequently found in the basal ganglia, thalamus and hypothalamus. In one of the most severely affected case, the NFT were even found in the neurons in the inferior olivary nucleus and in the spinal cord. The NFT were immunostained with Alz 50, and cosisted of paired helical filaments. The distribution of the neurons bearing the NFT was generally similar to that of the swollen storage neurons, and storage neurons often contained NFT in their perikarya and/or in the meganeurites. However, neurons with NFT could be noted without swollen perikarya. The coexistence of neuronal storage and NFT in NPC without amyloid deposits suggests that perturbed cholesterol metabolism and/or lysosomal membrane trafficking may play a role in the formation of NFT, and that amyloid deposits are not necessarily the prerequisite for NFT formation. The results of our study also suggest that NFT formation may be a rather nonspecific cellular reaction of neurons to certain slowly progressive metabolic perturbations of an as yet undefined nature.Part of this study was presented at the Annual Meeting of the Canadian Association of Neuropathologists, Whistler, BC, Canada, September 1993     

Hippocampal neurons predisposed to neurofibrillary tangle formation are enriched in type II calcium/calmodulin-dependent protein kinase

The microtubule-associated phosphoprotein, tau, is an integral component of paired helical filaments in Alzheimer neurofibrillary tangles (NFT). The mechanism of NFT formation is unknown but aberrant phosphorylation of tau may be contributory. Calcium/calmodulin-dependent protein kinase type II (CaM kinase II), the most abundant kinase in the brain, phosphorylates tau in vitro. We found CaM kinase II immunoreactivity concentrated in human hippocampal pyramidal neurons of CA1 and the subiculum. In Alzheimer's disease (AD) staining intensity of CA1 and subicular neurons is strikingly increased despite NFT formation and neuronal depletion. Enhanced CaM kinase II activity, possibly a result of deafferentation, may contribute to phosphorylation of tau protein leading to NFT deposition and neuronal death in AD.     

Overexpression of four-repeat tau mRNA isoforms in progressive supranuclear palsy but not in Alzheimer's disease.

Perturbations in the microtubule-associated protein tau occur in several human neurodegenerative diseases. In Alzheimer's disease and progressive supranuclear palsy (PSP), tau proteins assemble into straight and paired helical filaments that form intraneuronal deposits of neurofibrillary tangles (NFTs). The mechanisms underlying the aberrant assembly of tau into NFTs is unknown. To determine whether alterations in the expression of the carboxyl-terminal variants of tau contribute to NFT formation, we analyzed tau mRNA isoform expression in select regions of control, Alzheimer's disease, and PSP brains. In Alzheimer's disease, there were no alterations in tau mRNA isoform expression. However, in PSP, the levels of tau mRNA isoforms containing four microtubule binding domains were increased in the brainstem but not the frontal cortex or cerebellum. The brainstem in PSP has extensive NFT pathology, whereas the frontal cortex and cerebellum are relatively spared, suggesting that alterations in tau mRNA isoform expression occur in NFT-vulnerable regions in this disease. An increase in the four-repeat tau mRNA may lead to an increase in four-repeat tau protein isoforms and may contribute to the formation of NFTs in PSP. A similar increase in four-repeat tau mRNA has been reported for mutations associated with frontotemporal dementia and parkinsonism linked to chromosome 17.     

It may take inflammation, phosphorylation and ubiquitination to 'tangle' in Alzheimer's disease

Neurofibrillary tangles (NFT) are one of the pathologic hallmarks of Alzheimer's disease (AD). Their major component is tau, a protein that becomes hyperphosphorylated and accumulates into insoluble paired helical filaments. During the course of the disease such filaments aggregate into bulky NFT that get ubiquitinated. What triggers their formation is not known, but neuroinflammation could play a role. Neuroinflammation is an active process detectable in the earliest stages of AD. The neuronal toxicity associated with inflammation makes it a potential risk factor in the pathogenesis of chronic neurodegenerative diseases, such as AD. Determining the sequence of events that lead to this devastating disease has become one of the most important goals for AD prevention and treatment. In this review we focus on three topics relevant to AD pathology and to NFT formation: (1) what triggers CNS inflammation resulting in glia activation and neuronal toxicity; (2) how products of inflammation might change the substrate specificity of kinases/phosphatases leading to tau phosphorylation at pathological sites; (3) the relationship between the ubiquitin/proteasome pathway and tau ubiquitination and accumulation in NFT. The overall aim of this review is to provide a challenging and sometimes provocative survey of important contributions supporting the view that CNS inflammation might be a critical contributor to AD pathology. Neuronal cell death resulting from neuroinflammatory processes may have devastating effects as, in the vast majority of cases, neurons lost to disease cannot be replaced. In order to design therapies that will prevent endangered neurons from dying, it is critical that we learn more about the effects of neuroinflammation and its products.     

Relevance of neuronal and glial NPC1 for synaptic input to cerebellar Purkinje cells

Niemann–Pick type C disease is a rare and ultimately fatal lysosomal storage disorder with variable neurologic symptoms. The disease-causing mutations concern NPC1 or NPC2, whose dysfunction entails accumulation of cholesterol in the endosomal–lysosomal system and the selective death of specific neurons, namely cerebellar Purkinje cells. Here, we investigated whether neurodegeneration is preceded by an imbalance of synaptic input to Purkinje cells and whether neuronal or glial absence of NPC1 has different impacts on synapses. To this end, we prepared primary cerebellar cultures from wildtype or NPC1-deficient mice that are glia-free and highly enriched with Purkinje cells. We report that lack of NPC1 in either neurons or glial cells did not affect the excitability of Purkinje cells, the formation of dendrites or their excitatory synaptic activity. However, simultaneous absence of NPC1 from neuronal and glial cells impaired the presynaptic input to Purkinje cells suggesting a cooperative effect of neuronal and glial NPC1 on synapses.     

The early induction of cyclooxygenase 2 associated with neurofibrillary degeneration in brains of patients with Fukuyama-type congenital muscular dystrophy

Brains of thirteen patients with Fukuyama-type congenital muscular dystrophy (FCMD) were evaluated regarding the expression of cyclooxygenase 2 (COX2), an enzyme involved in the synthetic pathway of prostaglandins and thromboxanes, as well as neurofibrillary tangles (NFT). The neuronal induction of COX2 was demonstrated with immunohistochemistry and Western blotting confirmed the up-regulation. Preceded by COX2 immunoreactivity, NFT-containing neurons appeared in the majority of FCMD patients without beta-amyloid deposition or senile plaques. The hippocampus did not demonstrate neurodegeneration, while, in other areas, neurons with NFT spread in a similar manner to Alzheimer's disease. NFT-bearing neurons were concomitantly shown to be immunoreactive to COX2. The precedent induction of COX2, therefore, may be related to the formation of NFT in this genetic disorder.     

Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum

NeuN is a 46/48-kD nuclear protein antigen used widely to identify postmitotic neurons in both research and diagnostics. It is expressed by neurons throughout the nervous system of a variety of species, including birds, rodents, and man (Mullen et al. [1992] Development 116:201-211). When we sought to use NeuN to follow the developmental progression of murine cerebellar interneurons, we observed that expression of this antigen in the cerebellum was restricted to granule neurons and a small population of cells present in the lower molecular layer of the adult cerebellum. In an attempt to identify these cells, we combined immunostaining for NeuN with a panel of cell type-specific markers to unambiguously identify neurons that express NeuN in the adult and developing cerebellum. In contrast to postmitotic granule neurons, NeuN was not expressed by any other immunocytochemically identified cerebellar interneurons, which comprised basket and stellate cells, Golgi neurons, unipolar brush cells, and Lugaro cells. NeuN-positive cells in the molecular layer failed to express any cell type-specific markers tested. They may represent ectopic granule cells; alternatively, they may represent a hitherto unknown population of cerebellar cells. In vitro experiments suggest that NeuN expression is related closely to granule cell axogenesis. This approach also revealed that the level of NeuN expression could be modulated by chronically depolarizing these cells. Thus, whereas NeuN expression per se is a reliable marker of proliferative capacity, levels of NeuN expression may also be indicative of the physiological status of a postmitotic neuron.     

Accumulation of cyclin-dependent kinase 5 (cdk5) in neurons with early stages of Alzheimer''s disease neurofibrillary degeneration

Cyclin-dependent kinase 5 (cdk5) is one of the candidate kinases involved in the abnormal hyperphosphorylation of tau. To have a direct effect on tau hyperphosphorylation, cdk5 protein levels and enzyme activity should be upregulated in especially those neurons that develop neurofibrillary tangles (NFTs). We studied the distribution of cdk5 immunoreactivity in neurons with or without early- and late-stage NFTs in hippocampal, entorhinal, transentorhinal, temporal and frontal cortices, and cerebellum of Alzheimer's disease (AD) and control brain. The immunocytochemical localisation of cdk5 was compared with that obtained using antibodies to PHF-tau (tau in paired helical filaments of NFTs, mAb AT8) and ubiquitin as markers of early and late stage NFTs, respectively. Immunoreactivities of cdk5 and PHF-tau were found in neuronal perikarya and processes of hippocampal, entorhinal, transentorhinal, temporal and frontal, and cerebellar cortices. An apparent increase of cdk5 immunoreactivity was seen in pretangle neurons and in neurons bearing early stage NFTs. These findings suggest that this kinase might be involved in the formation of NFTs at a relatively early stage in the neocortex.     

Divergent phosphorylation pattern of tau in P301L tau transgenic mice

Aggregates of hyperphosphorylated tau are prominent in brains of patients with Alzheimer's disease or frontotemporal dementia (FTD). They have been reproduced in animal models following the identification of tau mutations in familial cases of FTD. This includes our previously generated transgenic model, pR5, which expresses FTD (P301L) mutant tau in neurons. The mice are characterized by tau aggregation including tangle (NFT) formation, memory impairment and mitochondrial dysfunction. In 8-month-old mice, S422 phosphorylation of tau is linked to NFT formation, however, a detailed analysis of tau solubility, phosphorylation and aggregation has not been done nor have the mice been monitored until a high age. Here, we undertook an analysis by immunohistochemistry, Gallyas impregnation and Western blotting of brains from 3 month- up to 20 month-old mice. NFTs first appeared at 6 months in the amygdala, followed by the CA1 region of the hippocampus. As the mice get older, the solubility of tau is decreased as determined by sequential extractions. Histological analysis revealed increased phosphorylation at the AT180, AT270 and 12E8 epitopes with ageing. The numbers of AT8-positive neurons increased from 3 to 6 months old. However, whereas S422 appeared only late and concomitantly with NFT formation, the only neurons left with AT8-reactivity at 20 months were those that had undergone NFT formation. As hyperphosphorylated tau continued to accumulate, the lack of AT8-reactivity suggests regulatory mechanisms in specifically dephosphorylating the AT8 epitope in the remaining neurons. Thus, differential regulation of phosphorylation is important for NFT formation in neurodegenerative diseases with tau pathology.     

Oxidative stress differentially induces tau dissociation from neuronal microtubules in neurites of neurons cultured from different regions of the embryonic Gallus domesticus brain

Abnormal phosphorylation of microtubule-associated proteins such as tau has been shown to play a role in neurodegenerative disorders. It is hypothesized that oxidative stress-induced aggregates of hyperphosphorylated tau could lead to the microtubule network degradation commonly associated with neurodegeneration. We investigated whether oxidative stress induced tau hyperphosphorylation and focused on neurite degradation using cultured neurons isolated from the embryonic chick brain as a model system. Cells were isolated from the cerebrum, cerebellum, and tectum of 14-day-old chicks, grown separately in culture, and treated with tert-Butyl hydroperoxide (to simulate oxidative stress) for 48 hr. Relative expression and localization of tau or phospho-tau and β-tubulin III in neurites were determined using quantitative immunocytochemistry and confocal microscopy. In untreated cells, tau was tightly colocalized with β-tubulin III. Increasing levels of oxidative stress induced an increase in overall tau expression in neurites of cerebral and tectal but not the cerebellar neurons, coupled with a decrease in phospho-tau expression in tectal but not the cerebral or cerebellar neurons. In addition, oxidative stress induced the degeneration of the distal ends of the neurites and redistribution of phospho-tau toward the neuronal soma in the cerebral but not the tectal and cerebellar neurons. These results suggest that oxidative stress induces changes in tau protein that precede cytoskeletal degradation and neurite retraction. Additionally, there is a differential susceptibility of neuronal subpopulations to oxidative stress, which may offer potential avenues for investigation of the cellular mechanisms underlying the differential manifestations of neurodegenerative disorders in different regions of the brain.     

Cdk5 and munc-18/p67 co-localization in early stage neurofibrillary tangles-bearing neurons in Alzheimer type dementia brains

Hyperphosphorylation of tau protein occurs during the formation of paired helical filament (PHF) in the brain with Alzheimer's disease. As previously reported, cyclin-dependent kinase (cdk) 5 can phosphorylate tau at the site of abnormally phosphorylated in PHF. To characterize the relationship between cdk5 and PHF-tau, we investigated the localization of cdk5 and its regulator, p67 (munc 18), in the hippocampus and temporal lobes from 12 Alzheimer type dementia (ATD) patients and 5 controls using immunohistochemical procedures. The specificity of antibodies was confirmed with Western blot analysis. Anti-cdk5 antibody diffusely stained the perikarya of some tau2-positive or neurofibrillary tangle (NFT)-bearing neurons in ATD brains, while cdk5-positive staining was scarcely found in control brains. Anti-p67 antibody also showed stronger immunoreactivity of pyramidal neurons in ATD brains than in control brains. Double immunostaining with anti-cdk5 and anti-p67 antibodies revealed co-localization of both molecules in some pyramidal neurons. These findings suggest that cdk5 is activated by p67 at the early stage of NFT formation and accelerates NFT formation. In cdk5-positive and p67-negative neurons, cdk5 may be activated by other regulator molecules such as p35. In addition, cdk5-positive reactive astrocytes were found close to cdk5-positive NFT-bearing neurons m ATD brains but not in control brains, suggesting a correlation between NFT and reactive astrocytes.     

Relationship between neuronal loss and tangle formation in neurons and oligodendroglia in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a progressive degenerative disorder characterized by neuronal loss, gliosis and abnormal fibril formation of abnormally phosphorylated tau protein in neurons and glia cells, but the cause is not clear at present. For the purpose of clarifying the pathological significance of accumulation of tau protein in neurons and oligodendroglia in PSP, we morphologically classified neurofibrillary tangles (NFT) and coiled bodies (CB) in oligodendroglia in three PSP brains into four stages, using double staining for immunohistochemistry with AT8 antibody and modified Gallyas‐Braak (GB) staining. AT8‐positive neurons without abnormal fibril structure with GB staining were classified as stage I, AT8‐positive neurons containing a few fibril structures with GB staining were classified stage II, AT8‐positive neurons containing mature fibril structures were classified as stage III, and AT8 negative neurons containing abnormal fibril structures stained only with GB staining were classified as stage IV (ghost tangles). These stages were also assessed for CB. Then we counted the number of cells of each stage in various brain regions to investigate the relationship of NFT and CB with neuronal loss and gliosis. The results showed that there were very few stage IV NFT and CB, which reflect cell death, but that stage III NFT and CB were abundant. Moreover, CB were abundant in regions with severe neuronal loss. These results suggest that appearance of CB is closely associated with degenerative regions.     

Increased level of active GSK-3beta in Alzheimer's disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration

The somatodendritic accumulation of hyperphosphorylated tau proteins is an early event preceding the appearance of neurofibrillary tangles (NFT) in Alzheimer's disease (AD) and might be necessary for their formation. Glycogen synthase kinase-3beta (GSK-3beta) is a physiological kinase for tau that generates many tau phosphorylation sites identified in NFT and in other tau-positive inclusions. We have studied the cellular distribution and the expression of the active form of GSK-3beta (GSK-3 pTyr216) in AD patients, in argyrophilic grain disease and in diffuse Lewy body disease. By Western blotting analysis, a significant increase in the level of GSK-3 (pTyr216) was observed in the frontal cortex of AD patients. A population of neurones showed a somatodendritic accumulation of GSK-3 (pTyr216) but not of the inactive form of GSK-3beta (GSK-3 pSer9). Most of these GSK-3 (pTyr216)-positive cells were positive for six different phosphotau epitopes known to be generated by GSK-3beta. By using a quadruple labelling method using GSK-3 (pTyr216) and phosphotau immunolabelling combined with Gallyas and DAPI staining, we examined neurones containing a somatodendritic GSK-3 (pTyr216) immunoreactivity at different stages of neurodegeneration. A majority of neurones at the pretangle stage without Gallyas-positive inclusions were GSK-3 (pTyr216) positive and this GSK-3 (pTyr216) immunoreactivity remained in most cells containing Gallyas and phosphotau-positive inclusions excepted in extracellular NFT. A GSK-3 (pTyr216) immunoreactivity was present in argyrophilic grains but not in cortical Lewy bodies. These results directly suggest that the activity of GSK-3beta is increased in AD and that somatodendritic accumulation and activation of GSK-3beta is an early event preceding and accompanying the formation of NFT and of other tau-positive inclusions.     

Neurons may live for decades with neurofibrillary tangles

Neurons containing neurofibrillary tangles (NFT) are one of the pathological hallmarks of Alzheimer disease (AD). It is known that this population of neurons express gene products and thus function to some degree, but it is unknown how long these neurons may survive with NFT. It is also thought that the formation of NFT results in the death of neurons. Using quantitative data on neuron loss and NFT formation as a function of disease duration, we have generated a computer program that models both the degeneration of CA1 hippocampal neurons and the formation of NFT in these neurons in AD. Modeling various neuron survival times with NFT and altering selected assumptions upon which the models are based, we arrive at the conclusions that 1) CA1 hippocampal neurons survive with NFT for about 20 years, and 2) NFT may not be obligatory for death of CA1 hippocampal neurons in AD.     

Molecular chaperone-mediated tau protein metabolism counteracts the formation of granular tau oligomers in human brain

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases termed tauopathies. The pathogenesis of tauopathies remains largely unknown. Molecular chaperones such as heat shock proteins (HSPs), however, have been implicated in tauopathies as well as in other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates. To search for in vivo evidence of chaperone-related tau protein metabolism, we analyzed human brains with varying degrees of neurofibrillary tangle (NFT) pathology, as defined by Braak NFT staging. Quantitative analysis of soluble protein levels revealed significant positive correlations between tau and Hsp90, Hsp40, Hsp27, alpha-crystallin, and CHIP. An inverse correlation was observed between the levels of HSPs in each specimen and the levels of granular tau oligomers, the latter of which were isolated from brain as intermediates of tau filaments. We speculate that HSPs function as regulators of soluble tau protein levels, and, once the capacity of this chaperone system is saturated, granular tau oligomers form virtually unabated. This is expressed pathologically as an early sign of NFT formation. The molecular basis of chaperone-mediated protection against neurodegeneration might lead to the development of therapeutics for tauopathies. (c) 2007 Wiley-Liss, Inc.     

Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice

Senile plaques comprised of Aβ aggregates and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments are the hallmarks of Alzheimer’s disease (AD). A number of amyloid precursor protein (APP) transgenic (Tg) mice harboring APP mutations have been generated as animal models of AD. These mice successfully display amyloid plaque formation and subsequent tau hyperphosphorylation, but seldom induce NFT formations. We have demonstrated that the APP_OSK-Tg mice, which possess the E693Δ (Osaka) mutation in APP and thereby accumulate Aβ oligomers without plaques, exhibit tau hyperphosphorylation at 8 months, but not NFT formation even at 24 months. We assumed that APP-Tg mice, including ours, failed to form NFTs because NFT formation requires human tau. To test this hypothesis, we crossbred APP_OSK-Tg mice with tau-Tg mice (tau264), which express low levels of 3-repeat and 4-repeat wild-type human tau without any pathology. The resultant double Tg mice displayed tau hyperphosphorylation at 6 months and NFT formation at 18 months in the absence of tau mutations. Importantly, these NFTs contained both 3-repeat and 4-repeat human tau, similar to those in AD. Furthermore, the double Tg mice exhibited Aβ oligomer accumulation, synapse loss, and memory impairment at 6 months and neuronal loss at 18 months, all of which appeared earlier than in the parent APP_OSK-Tg mice. These results suggest that Aβ and human tau synergistically interact to accelerate each other’s pathology, that the presence of human tau is critical for NFT formation, and that Aβ oligomers can induce NFTs in the absence of amyloid plaques.