Response of cell cycle proteins to neurotrophic factor and chemokine stimulation in human neuroglia

Increased expression of neurotrophins (e.g., NGF, BDNF) and chemokines (e.g., RANTES) has been observed in neurodegenerative diseases. We examined the effect of these factors on intracellular signaling cascades inducing cell cycle proteins p53, pRb, and E2F1 in human fetal mixed neuronal and glial cells. Comparing neurotrophin- and chemokine-treated cultures with untreated controls showed altered subcellular localization and expression of hyperphosphorylated retinoblastoma protein (ppRb), E2F1, and p53. Using immunofluorescent laser confocal microscopy, E2F1 and ppRb were detected exclusively in neuronal nuclei in control cultures while p53 was cytoplasmic in astrocytes and nuclear in neurons. Following treatment with neurotrophins, E2F1 and ppRb were observed in the cytoplasm of neurons, while p53 was observed in both neuronal and astrocytic nuclei. Similar findings were observed following treatment with RANTES. Semiquantitative analysis using immunoblots showed an increase in the amount of phosphorylated pRb in treated cultures. Induction of cell cycle proteins may play a role in neurodegeneration associated with neurotrophin and chemokine stimulation.     

Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein

Neurofibrillary tangles (NFTs) are a pathological hallmark of many neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). However, the cellular origin and the consequence of the NFT formation are poorly understood. Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the pathogenesis of sporadic AD, which accounts for the majority of AD occurrence. Here we show that after a transient cerebral ischemia, hyperphosphorylated tau accumulates in cortical neurons in a site-specific manner. The hyperphosphorylated tau presents a conformation similar to those present in human tauopathies, and colocalizes largely with signs of apoptosis. Our current study suggests that tau hyperphosphorylation may contribute to the brain damage induced by transient cerebral ischemia, and may be involved in the pathogenesis of neurodegenerative disorders in patients after stroke. Further, these results indicate that ischemic neuronal damage and apoptosis associates with tau hyperphosphorylation, and potentially NFTs formation. Finally, our results also suggest that neuronal apoptosis may be a therapeutic target in preventing tauopathy-related neurodegenerative diseases.     

Neuronal COX-2 expression and phosphorylation of pRb precede p38 MAPK activation and neurofibrillary changes in AD temporal cortex

In Alzheimer's disease (AD) brain, increased levels of cyclooxygenase-2 (COX-2), cell cycle markers, and p38 MAP kinase (MAPK) can be detected in neuronal cells. Besides mediating COX-2 expression, p38 MAPK is suggested to mediate cell cycle progression through phosphorylation of the retinoblastoma protein (pRb). In this study, we show that neuronal immunoreactivity for phosphorylated p38 MAPK does not correlate with COX-2 or phosphorylated pRb (ppRb) in control and AD temporal cortex. Immunoreactivity for activated p38 MAPK co-localizes with AT8 immunoreactivity and increases with the occurrence of neurofibrillary tangles and plaques. On the other hand, COX-2 immunoreactivity co-localizes and correlates with ppRb immunoreactivity in pyramidal neurons. COX-2 and ppRb do not co-localize with AT8 and decrease with increasing pathology. These results suggest that p38 MAPK does not mediate COX-2 expression and pRb inactivation, which are involved in cellular changes in pyramidal neurons early in AD pathogenesis.     

Proteasome inhibition induces stress kinase dependent transport deficits — Implications for Alzheimer's disease

Alzheimer's disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD.     

DJ-1 (PARK7) is associated with 3R and 4R tau neuronal and glial inclusions in neurodegenerative disorders

Mutations in the DJ-1 gene are associated with autosomal recessive Parkinson's disease (PD), but its role in disease pathogenesis is unknown. This study examines DJ-1 immunoreactivity (DJ-1 IR) in a variety of neurodegenerative disorders, Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) with Pick bodies, FTLD with MAPT mutations, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), in which hyperphosphorylated tau inclusions are the major pathological signature. DJ-1 IR was seen in a subset of neurofibrillary tangles (NFTs), neuropil threads (NTs), and neurites in extracellular plaques in AD; tau inclusions in AD contained both 3R and 4R tau. A subset of Pick bodies in FTLD showed DJ-1 IR. In PSP, DJ-1 IR was present in a few NFTs, NTs and glial cell inclusions. In CBD, DJ-1 IR was seen only in astrocytic plaques. In cases of FTLD with MAPT mutations that were 4R tau positive (i.e. N279K and exon 10+16 mutations), DJ-1 IR was present mostly in oligodendroglial coiled bodies. However, in MAPT R406W mutation cases, DJ-1 IR was associated mainly with NFTs and NTs and these were both 3R and 4R tau positive. No DJ-1 IR was present in FTLD with ubiquitin inclusions (FTLD-U). In AD and FTLD with Pick bodies, DJ-1 protein was enriched in the sarkosyl-insoluble fractions of frozen brain tissue containing insoluble hyperphosphorylated tau, thus strengthening the association of DJ-1 with tau pathology. Additionally using two-dimensional gel electrophoresis, we demonstrated accumulation of acidic pI isoforms of DJ-1 in AD brain, which may compromise its normal function. Our observations confirm previous findings that DJ-1 is present in a subpopulation of glial and neuronal tau inclusions in tau diseases and associated with both 3R and 4R tau isoforms.     

Tau and transgenic animal models

Advances in genetics and transgenic approaches have a continuous impact on our understanding of Alzheimer’s disease (AD) and related disorders, especially as aspects of the histopathology and neurodegeneration can be reproduced in animal models. AD is characterized by extracellular Aβ peptide-containing plaques and neurofibrillary aggregates of hyperphosphorylated isoforms of microtubule-associated protein tau. A causal link between Aβ production, neurodegeneration and dementia has been established with the identification of familial forms of AD which are linked to mutations in the amyloid precursor protein APP, from which the Aβ peptide is derived by proteolysis. No mutations have been identified in the tau gene in AD until today. Tau filament formation, in the absence of Aβ production, is also a feature of several additional neurodegenerative diseases including progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). The identification of mutations in the tau gene which are linked to FTDP-17 established that dysfunction of tau can, as well as Aβ formation, lead to neurodegeneration and dementia. In this review, newly recognized cellular functions of tau, and the neuropathology and clinical syndrome of FTDP-17 will be presented, as well as recent advances that have been achieved in studies of transgenic mice expressing tau and AD-related kinases and phosphatases. These models link neurofibrillary lesion formation to neuronal loss, provide an in vivo model in which therapies can be assessed, and may contribute to determine the relationship between Aβ production and tau pathology.     

Tau and tauopathies

Tau protein is a neuronal microtubule-associated protein (MAP), which localizes primarily in the axon. It is one of the major and most widely distributed MAPs in the central nervous system. Its biochemistry and molecular pathology is being increasingly studied. Tau is a key component of neurofbrillary tangles in Alzheimer's disease (AD). Disorders with neuronal, oligodendroglial or astrocytic filamentous tau inclusions are now grouped under the common rubric of tauopathies. The discovery of mutations in the tau gene, located on Chromosome 17 and its relationship to frontotemporal dementia with Parkinsonism (FTDP-17) has enhanced the importance of tau protein in cognitive neurology. Aberrant aggregates of tau have been documented in most of the neurodegenerative diseases with filamentous inclusions. The role of cerebrospinal fluid tau in the diagnosis of dementias is being investigated quite extensively. Recently, it has been shown that Abeta immunotherapy leads to the clearance of early tau pathology. It is becoming clearer that understanding tau better will lead to better understanding of many neurodegenerative diseases that may help develop interventional strategies.     

Amyloid deposition in APP23 mice is associated with the expression of cyclins in astrocytes but not in neurons

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the intracellular accumulation of highly phosphorylated tau protein, the extracellular formation of amyloid plaques and a significant loss of neurons. Recent evidence suggests that neuronal death in AD involves an aborted attempt of cells to re-enter the cell cycle. To study the effect of amyloid deposits on cell cycle related events in vivo, the expression of cell cycle markers was examined by immunohistochemistry in amyloid precursor protein (APP) transgenic mice (APP23 mice, Swedish double mutation). A deposition in APP23 mice is associated with prominent gliosis that is characterized by an astrocytic expression of cyclins D1, E and B1 as well as the nuclear translocation of cyclin-dependent protein kinase 4. However, amyloid plaque formation is not accompanied by significant changes in the neuronal expression of cyclins or cyclin-dependent kinase inhibitors. It is concluded, therefore, that in contrast to AD, amyloid pathology in APP23 mice is not associated with changes in the expression of cell cycle markers in neurons. The results support the assumption that the neuronal re-expression of cell cycle components in AD is not a consequence of A formation and deposition.     

Clinical proteomics in neurodegenerative disorders

Neurodegenerative disorders are characterized by neuronal impairment that eventually leads to neuronal death. In spite of the brain's known capacity for regeneration, lost neurons are difficult to replace. Therefore, drugs aimed at inhibiting neurodegenerative processes are likely to be most effective if the treatment is initiated as early as possible. However, clinical manifestations in early disease stages are often numerous, subtle and difficult to diagnose. This is where biomarkers that specifically reflect onset of pathology, directly or indirectly, may have a profound impact on diagnosis making in the future. A triplet of biomarkers for Alzheimer's disease (AD), total and hyperphosphorylated tau and the 42 amino acid isoform of β-amyloid, has already been established for early detection of AD before the onset of dementia. However, more biomarkers are needed both for AD and for other neurodegenerative disorders, such as Parkinson's disease, frontotemporal dementia and amyotrophic lateral sclerosis. This review provides an update on recent advances in clinical neuroproteomics, a biomarker discovery field that has expanded immensely during the last decade, and gives an overview of the most commonly used techniques and the major clinically relevant findings these techniques have lead to.     

Olfactory bulb involvement in neurodegenerative diseases

Olfactory dysfunction is a common and early symptom of many neurodegenerative diseases, particularly of Parkinson’s disease and other synucleinopathies, Alzheimer’s disease (AD), and mild cognitive impairment heralding its progression to dementia. The neuropathologic changes of olfactory dysfunction in neurodegenerative diseases may involve the olfactory epithelium, olfactory bulb/tract, primary olfactory cortices, and their secondary targets. Olfactory dysfunction is related to deposition of pathological proteins, α-synuclein, hyperphosphorylated tau protein, and neurofilament protein in these areas, featured by neurofibrillary tangles, Lewy bodies and neurites inducing a complex cascade of molecular processes including oxidative damage, neuroinflammation, and cytosolic disruption of cellular processes leading to cell death. Damage to cholinergic, serotonergic, and noradrenergic systems is likely involved, since such damage is most marked in those diseases with severe anosmia. Recent studies of olfactory dysfunction have focused its potential as an early biomarker for the diagnosis of neurodegenerative disorders and their disease progression. Here, we summarize the current knowledge on neuropathological and pathophysiological changes of the olfactory system in the most frequent neurodegenerative diseases, in particular AD and synucleinopathies. We also present neuropathological findings in the olfactory bulb and tract in a large autopsy cohort (n = 536, 57.8 % female, mean age 81.3 years). The severity of olfactory bulb HPτ, Aβ, and αSyn pathology correlated and increased significantly (P < 0.001) with increasing neuritic Braak stages, Thal Aβ phases, and cerebral Lewy body pathology, respectively. Hence, further studies are warranted to investigate the potential role of olfactory biopsies (possibly restricted to the olfactory epithelium) in the diagnostic process of neurodegenerative diseases in particular in clinical drug trials to identify subjects showing early, preclinical stages of neurodegeneration and to stratify clinically impaired cohorts according to the underlying cerebral neuropathology.     

The role of neurotrophins and insulin on tau pathology in Alzheimer's disease

Alterations in the structure and function of tau protein is the primary pathology of a variety of neurodegenerative diseases, including Alzheimer's disease (AD). In these diseases, hyperphosphorylated tau protein forms aggregates which are deposited in the somadendritic regions of the neurons in the central nervous system. This series of events is toxic to neurons, and plays a crucial role in disease development. However, the events leading to the deregulation of tau protein in AD are not clear. Recently, there has been much research into the possible roles of neurotrophic factors in AD. AD brain exhibits changes in levels of different neurotrophic factors, including brain-derived neurotrophic factor and nerve growth factor. These neurotrophic factors are known to be important for the proper functioning of neurons, and their deregulation may play an important role in AD disease progression. Of particular interest, these neurotrophic factors may play a role in the regulation and proper function of tau protein. In this review, the roles of neurotrophic factors in AD and in the regulation of tau protein are discussed.     

Coexisting pathologies in the brain: influence of vascular disease and Parkinson’s disease on Alzheimer’s pathology in the hippocampus

The finding of more than one coexisting brain pathology in dementia sufferers is not unusual. However, it is unclear how these different diseases may interact or influence the evolution of one another. In this study we analyse the hippocampal expression patterns of hyperphosphorylated tau, paired helical filament (PHF)-related protein, β-amyloid and synaptophysin in a group of Alzheimer’s disease (AD) sufferers with and without additional pathology. Compared to cases with only AD-type pathology we found that the presence of additional vascular disease augmented the accumulation of hyperphosphorylated tau in the CA1 region of the hippocampus without affecting PHF formation in cases with mild AD changes and reduced the extent of PHF formation in the CA2/3 and CA4 regions of the hippocampus in cases with severe AD pathology. We also found that synaptophysin immunoreactivity in the CA4 and dentate gyrus in pure AD was inversely related to the extent of amyloid accumulation but not to neurofibrillary pathology in the same regions. These relationships were lost when additional pathology was present. Memory scores obtained during life correlated closely with hyperphosphorylated tau and PHF-related protein expression in CA1 in pure AD but not in AD with additional pathology. Total amyloid and synaptophysin expression in the hippocampus did not correlate with memory scores in any patient group. Our findings suggest that the interactions of two pathologies in the hippocampus are complex and may differ depending on the stage reached in the evolution of a progressive disease such as AD. Received: 26 July 1999 / Revised, accepted: 12 October 1999     

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.     

Inhibition of proteasome and Shaggy/Glycogen synthase kinase-3beta kinase prevents clearance of phosphorylated tau in Drosophila

Tauopathies, including Alzheimer's disease (AD), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally phosphorylated tau protein. In AD brains, it has been shown that the level of abnormally phosphorylated tau is higher than in age-matched control brains, suggesting that abnormally phosphorylated tau is resistant to degradation. By using a Drosophila model of tauopathy, we studied the relationship between tau phosphorylation and degradation. We showed that in vivo reduction of proteasome activity results in an accumulation of high-molecular-weight forms of hyperphosphorylated tau. We also found that glycogen synthase kinase (GSK)-3beta-mediated hyperphosphorylated forms of tau are degradable by the proteasomal machinery. Unexpectedly, GSK-3beta inactivation resulted in a very large accumulation of high-molecular-weight species consisting of hyperphosphorylated tau, suggesting that, depending on the kinase(s) involved, tau phosphorylation state affects its degradation differently. We thus propose a model for tauopathies in which, depending on toxic challenges (e.g., oxidative stress, exposure to amyloid peptide, etc.), abnormal phosphorylation of tau by kinases distinct from GSK-3beta leads to progressive accumulation of hyperphosphorylated tau oligomers that are resistant to degradation.     

CSF biomarkers cutoffs: the importance of coincident neuropathological diseases

The effects of applying clinical versus neuropathological diagnosis and the inclusion of cases with coincident neuropathological diagnoses have not been assessed specifically when studying cerebrospinal fluid (CSF) biomarker classification cutoffs for patients with neurodegenerative diseases that cause dementia. Thus, 142 neuropathologically diagnosed neurodegenerative dementia patients [71 Alzheimer's disease (AD), 29 frontotemporal lobar degeneration (FTLD), 3 amyotrophic lateral sclerosis, 7 dementia with Lewy bodies, 32 of which cases also had coincident diagnoses] were studied. 96 % had enzyme-linked immunosorbant assay (ELISA) CSF data and 77 % had Luminex CSF data, with 43 and 46 controls for comparison, respectively. Aβ(42), total, and phosphorylated tau(181) were measured. Clinical and neuropathological diagnoses showed an 81.4 % overall agreement. Both assays showed high sensitivity and specificity to classify AD subjects against FTLD subjects and controls, and moderate sensitivity and specificity for classifying FTLD subjects against controls. However, among the cases with neuropathological diagnoses of AD plus another pathology (26.8 % of the sample), 69.4 % (ELISA) and 96.4 % (Luminex) were classified as AD according to their biomarker profiles. Use of clinical diagnosis instead of neuropathological diagnosis led to a 14-17 % underestimation of the biomarker accuracy. These results show that while CSF Aβ and tau assays are useful for diagnosis of AD and neurodegenerative diseases even at MCI stages, CSF diagnostic analyte panels that establish a positive diagnosis of Lewy body disease and FTLD are also needed, and must be established based on neuropathological rather than clinical diagnoses.     

Comparison of the neurofibrillary pathology in Alzheimer’s disease and familial presenile dementia with tangles

Alzheimer’s disease (AD) is characterised neuropathologically by the presence of abundant extracellular β-amyloid deposits and intracellular neurofibrillary lesions consisting of neurofibrillary tangles, neuropil threads and senile plaque neurites which contain paired helical filaments (PHFs) made of hyperphosphorylated microtubule-associated protein tau. A new familial form of presenile dementia with neurofibrillary pathology and no β-amyloid deposits has been described recently [Sumi et al. (1992) Neurology 42: 120–127]. We have compared the tau pathology in this familial form of presenile dementia with that of AD. To this end we have used electron microscopy, immunoblotting and immunohistochemistry with phosphorylation-dependent (PHF1, AT8, AT100, AT180, AT270, 12E8) and phosphorylation-independent (BR133, BR134) anti-tau antibodies. We show that in the two diseases dispersed PHFs are structurally, biochemically and immunologically identical; they are stained by all anti-tau antibodies used and on immunoblots PHF-tau appears as three major bands of 60, 64 and 68 kDa. However, while the anti-tau antibodies stain neurofibrillary tangles, neuropil threads and neuritic plaques in AD brain, no neuritic plaques are found in familial presenile dementia. These results indicate that in the two diseases tau undergoes the same modifications; they confirm that neurofibrillary tangles and neuropil threads like those in AD can exist independently of β-amyloid deposits and that their presence is associated with dementia.     

Alteraciones en la corteza motora primaria en la enfermedad de Alzheimer: estudio en el modelo 3xTg-AD

IntroductionIn humans and animal models, Alzheimer disease (AD) is characterised by accumulation of amyloid-β peptide (Aβ) and hyperphosphorylated tau protein, neuronal degeneration, and astrocytic gliosis, especially in vulnerable brain regions (hippocampus and cortex). These alterations are associated with cognitive impairment (loss of memory) and non-cognitive impairment (motor impairment). The purpose of this study was to identify cell changes (neurons and glial cells) and aggregation of Aβ and hyperphosphorylated tau protein in the primary motor cortex (M1) in 3xTg-AD mouse models at an intermediate stage of AD.MethodsWe used female 3xTg-AD mice aged 11 months and compared them to non-transgenic mice of the same age. In both groups, we assessed motor performance (open field test) and neuronal damage in M1 using specific markers: BAM10 (extracellular Aβ aggregates), tau 499 (hyperphosphorylated tau protein), GFAP (astrocytes), and Klüver-Barrera staining (neurons).ResultsFemale 3xTg-AD mice in intermediate stages of the disease displayed motor and cellular alterations associated with Aβ and hyperphosphorylated tau protein deposition in M1.ConclusionsPatients with AD display signs and symptoms of functional impairment from early stages. According to our results, M1 cell damage in intermediate-stage AD affects motor function, which is linked to progression of the disease.     

Notch-1 immunoexpression is increased in Alzheimer's and Pick's disease

Notch-1 is a protein that influences cell fate decisions, with its expression occurring primarily during embryogenesis and development. However, Notch-1 is also expressed in the adult brain, in regions with high synaptic plasticity, particularly the hippocampus. Its role in adults is unknown; however, it may impact neurite outgrowth or cell differentiation in adult brain regions undergoing neurogenesis. Notch-1 is increased in Alzheimer's disease (AD); however, its expression in other CNS degenerative diseases has not been described. To begin to define the range of degenerative disorders where Notch-1 expression is altered, we examined Notch-1 immunoreactivity in a variety of neurodegenerative diseases to determine whether its increase is selective for AD. We examined sections of hippocampus from 13 AD, 13 classical Pick's disease (PiD; with Pick bodies), 4 dementia lacking distinctive histopathology (DLDH) and 8 control brains, emphasizing hippocampal (dentate gyrus) pathology. We determined that Notch-1 immunoexpression is increased in AD and PiD relative to control cases. DLDH cases were not significantly different than control cases with respect to Notch-1 expression. Given the increase in Notch-1 immunoexpression in AD and PiD, two diseases where abnormal tau aggregates are present, and the lack of Notch-1 immunoexpression in DLDH (where tau aggregates are absent), we cannot rule out the possibility that tau aggregates are associated with Notch-1 expression in neurodegenerative diseases.