Tau is not normally degraded by the proteasome

Tau-positive inclusions in neurons are consistent neuropathologic features of the most common causes of dementias such Alzheimer's disease and frontotemporal dementia. Ubiquitinated tau-positive inclusions have been reported in brains of Alzheimer's disease patients, but involvement of the ubiquitin-dependent proteasomal system in tau degradation remains controversial. Before considering the tau degradation in pathologic conditions, it is important to determine whether or not endogenous tau is normally degraded by the proteasome pathway. We therefore investigated this question using two complementary approaches in vitro and in vivo. Firstly, SH-SY5Y human neuroblastoma cells were treated with different proteasome inhibitors, MG132, lactacystin, and epoxomicin. Under these conditions, neither total nor phosphorylated endogenous tau protein levels were increased. Instead, an unexpected decrease of tau protein was observed. Secondly, we took advantage of a temperature-sensitive mutant allele of the 20S proteasome in Drosophila. Genetic inactivation of the proteasome also resulted in a decrease of tau levels in Drosophila. These results obtained in vitro and in vivo demonstrate that endogenous tau is not normally degraded by the proteasome.     

A comparative study on pathological features of transgenic rat lines expressing either three or four repeat misfolded tau

Human tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by distinct clinical features, typical histopathological structures, and defined ratio(s) of three‐repeat and four‐repeat tau isoforms within pathological aggregates. How the optional microtubule‐binding repeat of tau influences this differentiation of pathologies is understudied. We have previously generated and characterized transgenic rodent models expressing human truncated tau aa151–391 with either three (SHR24) or four microtubule‐binding repeats (SHR72). Here, we compare the behavioral and neuropathological hallmarks of these two transgenic lines using a battery of tests for sensorimotor, cognitive, and neurological functions over the age range of 3.5–15 months. Progression of sensorimotor and neurological deficits was similar in both transgenic lines; however, the lifespan of transgenic line SHR72 expressing truncated four‐repeat tau was markedly shorter than SHR24. Moreover, the expression of three or four‐repeat tau induced distinct neurofibrillary pathology in these lines. Transgenic lines displayed different distribution of tau pathology and different type of neurofibrillary tangles. Our results suggest that three‐ and four‐repeat isoforms of tau may display different modes of action in the diseased brain.     

Review: Somatic mutations in neurodegeneration

Somatic mutations are postzygotic mutations which may lead to mosaicism, the presence of cells with genetic differences in an organism. Their role in cancer is well established, but detailed investigation in health and other diseases has only been recently possible. This has been empowered by the improvements of sequencing techniques, including single‐cell sequencing, which can still be error‐prone but is rapidly improving. Mosaicism appears relatively common in the human body, including the normal brain, probably arising in early development, but also potentially during ageing. In this review, we first discuss theoretical considerations and current evidence relevant to somatic mutations in the brain. We present a framework to explain how they may be integrated with current views on neurodegeneration, focusing mainly on sporadic late‐onset neurodegenerative diseases (Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis). We review the relevant studies so far, with the first evidence emerging in Alzheimer's in particular. We also discuss the role of mosaicism in inherited neurodegenerative disorders, particularly somatic instability of tandem repeats. We summarize existing views and data to present a model whereby the time of origin and spatial distribution of relevant somatic mutations, combined with any additional risk factors, may partly determine the development and onset age of sporadic neurodegenerative diseases.     

Synaptic alterations in the rTg4510 mouse model of tauopathy

Synapse loss, rather than the hallmark amyloid‐β (Aβ) plaques or tau‐filled neurofibrillary tangles (NFT), is considered the most predictive pathological feature associated with cognitive status in the Alzheimer's disease (AD) brain. The role of Aβ in synapse loss is well established, but despite data linking tau to synaptic function, the role of tau in synapse loss remains largely undetermined. Here we test the hypothesis that human mutant P301L tau overexpression in a mouse model (rTg4510) will lead to age‐dependent synaptic loss and dysfunction. Using array tomography and two methods of quantification (automated, threshold‐based counting and a manual stereology‐based technique) we demonstrate that overall synapse density is maintained in the neuropil, implicating synapse loss commensurate with the cortical atrophy known to occur in this model. Multiphoton in vivo imaging reveals close to 30% loss of apical dendritic spines of individual pyramidal neurons, suggesting these cells may be particularly vulnerable to tau‐induced degeneration. Postmortem, we confirm the presence of tau in dendritic spines of rTg4510‐YFP mouse brain by array tomography. These data implicate tau‐induced loss of a subset of synapses that may be accompanied by compensatory increases in other synaptic subtypes, thereby preserving overall synapse density. Biochemical fractionation of synaptosomes from rTg4510 brain demonstrates a significant decrease in expression of several synaptic proteins, suggesting a functional deficit of remaining synapses in the rTg4510 brain. Together, these data show morphological and biochemical synaptic consequences in response to tau overexpression in the rTg4510 mouse model. J. Comp. Neurol., 521:1334–1353, 2013. © 2012 Wiley Periodicals, Inc.     

Amyloid beta protein-related death-inducing protein induces G2/M arrest: Implications for neurodegeneration in Alzheimer's disease

Amyloid beta protein (Abeta)-related death-inducing protein (AB-DIP) is a novel Abeta binding protein expressed ubiquitously. Here we demonstrate that overexpression of AB-DIP in SH-SY5Y neuroblastoma cells causes G2/M arrest. By deletion mutant analysis, we have identified the minimal region within AB-DIP required for G2/M arrest. We have also shown that microtubule-interfering agents (MIAs) such as nocodazole, vinblastine, paclitaxel, and vincristine, known to arrest cells at G2/M, also phosphorylate AB-DIP. However, etoposide, which causes genotoxic stress; tunicamycin, an ER stress inducer; and rotenone, which causes mitochondrial damage, fail to phosphorylate AB-DIP, implying that phosphorylation of AB-DIP is specific to microtubule-disruption-induced G2/M arrest. By using different classes of kinase inhibitors, we also demonstrate that a putative tyrosine kinase phosphorylates AB-DIP. Mono- or multisite mutations of tyrosine or serine/threonine residues confirmed that mutation of tyrosine residues but not serine/threonine residues greatly reduces nocodazole-induced phosphorylation of AB-DIP. Furthermore, phosphorylation of AB-DIP can be induced in MCF-7 cells that lack functional p53, suggesting that AB-DIP phosphorylation is independent of p53. Mounting experimental evidence continues to support the role of cell cycle abnormalities in the pathogenesis of Alzheimer's disease, and our results suggest that AB-DIP might provide a mechanistic link between microtubule disruption, mitotic abnormalities, neuronal dysfunction, and death. Therefore, interfering with AB-DIP may have therapeutic applications in conditions such as Alzheimer's disease, in which microtubule disruption and mitotic abnormalities have been suggested to play a pathological role.     

Mounting evidence of FKBP12 implication in neurodegeneration

Intrinsically disordered proteins, such as tau or α-synuclein, have long been associated with a dysfunctional role in neurodegenerative diseases. In Alzheimer's and Parkinson's' diseases, these proteins, sharing a common chemical-physical pattern with alternating hydrophobic and hydrophilic domains rich in prolines, abnormally aggregate in tangles in the brain leading to progressive loss of neurons. In this review, we present an overview linking the studies on the implication of the peptidyl-prolyl isomerase domain of immunophilins, and notably FKBP12, to a variety of neurodegenerative diseases, focusing on the molecular origin of such a role. The involvement of FKBP12 dysregulation in the aberrant aggregation of disordered proteins pinpoints this protein as a possible therapeutic target and, at the same time, as a predictive biomarker for early diagnosis in neurodegeneration, calling for the development of reliable, fast and cost-effective detection methods in body fluids for community-based screening campaigns.     

Familial tauopathy with P364S MAPT mutation: clinical course,neuropathology and ultrastructure of neuronal tau inclusions

Aims

This report presents the clinical course, neuropathology and ultrastructure of neuronal tau inclusions of four Slovene relatives with P364S MAPT mutation.

Methods

The clinical history of three out of four P364S MAPT mutation carriers was taken. After formalin fixation, thorough sampling of the central nervous system was followed by paraffin embedding, H&E, Gallyas, Bielschowsky and immunostaining with AT8, anti‐3R, anti‐4R tau, anti‐amyloid‐β, anti‐TDP43 and anti‐alpha‐synuclein antibodies. The distribution and density of different types of neuronal tau inclusions were semiquantitatively assessed. In addition, the ultrastructure of neuronal tau inclusions was analysed.

Results

Macroscopic examination of the brains was unremarkable. Microscopically, neuronal tau inclusions of almost all known types were widespread and distributed fairly uniformly in all cases. Pick bodies and swollen neurones were found in only one family member. Mutant tau was composed of 3R and 4R isoforms, with a slight predominance of 3R tau. Composite neuronal tau inclusion (CNTI), found in all four relatives, was a hallmark of the P364S MAPT mutation. CNTI showed compartmental differences in H&E and Gallyas staining, tau isoforms immunolabelling and ultrastructure, displaying fuzzy fibrils in the core and paired twisted tubules at the periphery.

Conclusions

P364S MAPT mutation is characterized clinically by a variable combination of frontotemporal dementia, parkinsonism and motor neurone disease of short duration, and neuropathologically by a widespread uniform distribution of all known neuronal tau inclusions in one family member. Two‐compartment CNTI is a unique characteristic of the P364S MAPT mutation.     

Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual‐hit hypothesis of neurodegeneration

The dual‐hit hypothesis of neurodegeneration states that severe stress sensitizes vulnerable cells to subsequent challenges so that the two hits are synergistic in their toxic effects. Although the hippocampus is vulnerable to a number of neurodegenerative disorders, there are no models of synergistic cell death in hippocampal neurons in response to combined proteotoxic and oxidative stressors, the two major characteristics of these diseases. Therefore, a relatively high‐throughput dual‐hit model of stress synergy was developed in primary hippocampal neurons. In order to increase the rigor of the study and strengthen the interpretations, three independent, unbiased viability assays were employed at multiple timepoints. Stress synergy was elicited when hippocampal neurons were treated with the proteasome inhibitor MG132 followed by exposure to the oxidative toxicant paraquat, but only after 48 h. MG132 and paraquat only elicited additive effects 24 h after the final hit and even loss of heat shock protein 70 activity and glutathione did not promote stress synergy at this early timepoint. Dual hits of MG132 elicited modest glutathione loss and slightly synergistic toxic effects 48 h after the second hit, but only at some concentrations and only according to two viability assays (metabolic fitness and cytoskeletal integrity). The thiol N‐acetyl cysteine protected hippocampal neurons against dual MG132/MG132 hits but not dual MG132/paraquat hits. These findings support the view that proteotoxic and oxidative stress propel and propagate each other in hippocampal neurons, leading to synergistically toxic effects, but not as the default response and only after a delay. The neuronal stress synergy observed here lies in contrast to astrocytic responses to dual hits, because astrocytes that survive severe proteotoxic stress resist additional cell loss following second hits. In conclusion, a new model of hippocampal vulnerability was developed for the testing of therapies, because neuroprotective treatments that are effective against severe, synergistic stress are more likely to succeed in the clinic. © 2016 Wiley Periodicals, Inc.     

Absence of microglia or presence of peripherally-derived macrophages does not affect tau pathology in young or old hTau mice

Microglia are implicated in the pathophysiology of several neurodegenerative disorders, including Alzheimer's disease. While the role of microglia and peripheral macrophages in regulating amyloid beta pathology has been well characterized, the impact of these distinct cell subsets on tau pathology remains poorly understood. We and others have recently demonstrated that monocytes can engraft the brain and give rise to long-lived parenchymal macrophages, even under nonpathological conditions. We undertook the current study to investigate the regulation of tau pathology by microglia and peripheral macrophages using hTau transgenic mice, which do not exhibit microglial activation/pathology or macrophage engraftment. To assess the direct impact of microglia on tau pathology we developed a protocol for long-term microglial depletion in Cx3cr1^CreERR26^DTA mice and crossed them with hTau mice. We then depleted microglia up to 3 months in both young and old mice, but no net change in forebrain soluble oligomeric tau or total or phosphorylated levels of aggregated tau was recorded. To investigate the consequence of peripherally-derived parenchymal macrophages on tau aggregation we partially repopulated the hTau microglial pool with peripheral macrophages, but this also did not affect levels of tau oligomers or insoluble aggregates. Our study questions the direct involvement of microglia or peripheral macrophages in the development of tau pathology in the hTau model.     

Evidence of neurodegeneration in obstructive sleep apnea: Relationship between obstructive sleep apnea and cognitive dysfunction in the elderly

The incidence of dementia and obstructive sleep apnea (OSA) increases with age. Late‐onset Alzheimer's disease (AD) is an irreversible neurodegenerative disease of the elderly characterized by amyloid β (Aβ) plaques and neurofibrillary tangles. The disease involves widespread synaptic loss in the neocortex and the hippocampus. Rodent and clinical studies suggest that OSA impairs the structural integrity of several brain regions, including the medial temporal lobe. Indeed, hypoxia, hypertension, hypoperfusion, endothelial dysfunction, inflammation, and oxidative stress noted in OSA patients also occur in AD patients. This Review highlights pathological commonality, showing that OSA upregulates Aβ, tau hyperphosphorylation, and synaptic dysfunction. Indeed, OSA and hypertension trigger hypoperfusion and hypometabolism of brain regions, including cortex and hippocampus. Several studies show that hypertension‐driven brain damage and pathogenic mechanisms lead to an Aβ increase. The pathophysiological mechanism by which OSA enhances hypertension may be linked to sympathoexcitation, oxidative stress, and endothelial dysfunction. Strong pathophysiological similarities that exist between OSA and AD are underscored here. For example, the hippocampus is negatively impacted in both OSA and AD. OSA promotes hippocampal atrophy, which is associated with memory impairment. Cognitive impairment, even in the absence of manifest dementia, is an important independent predictor of mortality. However, several pathophysiological mechanisms in OSA are reversible with appropriate therapy. OSA, therefore, is a modifiable risk factor of cognitive dysfunction, and treating OSA prior to mild cognitive impairment may be an effective prevention strategy to reduce risk for cognitive decline and AD in middle‐aged persons and the elderly. © 2015 Wiley Periodicals, Inc.     

Loss of presenilin function causes Alzheimer's disease-like neurodegeneration in the mouse

Accumulating evidence has indicated that gain-of-function in beta-amyloid production may be not the necessary mechanism for mutant presenilin-1 (PS1) or PS2 to cause familial Alzheimer's disease (AD). In the present article, we show that conditional knockout of PS1 from the adult stage in the forebrain of mice with the PS2 null mutation triggers robust AD-like neurodegeneration including brain shrinkage, cortical and hippocampal atrophy,ventricular enlargement, severe neuronal loss, gliosis, tau hyperphosphorylation, neurofillament tangle-like structures, and intracellular filaments. Learning and memory functions in these mice are almost completely lost. Notably, there is no beta-amyloid deposition, indicating that presenilin dysfunction can directly cause neurodegeneration without the involvement of beta-amyloid. Furthermore, neurodegeneration occurs in a progressive manner following aging, suggesting that an accumulating effect of presenilin dysfunction over time might be a pathogenic mechanism for the involvement of mutant PS1/PS2 in causing AD. These results validate a mouse model characterized by the presence of many features of AD pathology. Furthermore, the demonstration of AD-like neurodegeneration in the absence of beta-amyloid deposition challenges the long-standing beta-amyloid cascade hypothesis and encourages an open debate on the role of beta-amyloid in causing AD. Most important, our results strongly suggest that to develop gamma-secretase inhibitors for the pharmacological treatment of AD may be not a reasonable strategy because antagonism of presenilin function may worsen neurodegeneration.     

Biochemistry and molecular biology of tauopathies

Filamentous tau deposits in neurons or glial cells are the hallmark lesions of neurodegenerative tauopathies, such as Alzheimer’s disease, Pick’s disease, corticobasal degeneration and progressive supranuclear palsy. Biochemical analyses of Sarkosyl‐insoluble tau from brains with tauopathies have revealed that tau deposits in different diseases consisted of different tau isoforms (i.e., all six tau isoforms occur in Alzheimer’s disease, four repeat tau isoforms occur in corticobasal degeneration or progressive supranuclear palsy, and three repeat tau isoforms occur in Pick’s disease). The discovery of mutations in the tau gene in FTDP‐17 has established that abnormalities in tau function or expression are sufficient to cause filamentous aggregation of hyperphosphorylated tau and neurodegeneration similar to that seen in sporadic tauopathies. Because the number of tau inclusions and their regional distribution correlate with clinical symptoms, inhibition of tau aggregation or filament formation in neurons or glial cells may prevent neurodegeneration. We have investigated the effects of 42 compounds belonging to nine different chemical classes on tau filament formation, and found that several phenothiazine and polyphenol compounds, and one porphyrin compound inhibit tau filament formation.     

Presence of axonal paried helical filament-tau in Alzheimer's disease: Submicroscopic localization

While normal adult tau protein is found typically in axons, paired helical filament (PHF) tau has been shown immunohistochemically to be in the somatodendritic compartment in Alzheimer's disease (AD). The small amount of PHF-tau (8--11%) found in white matter with immunochemical methods is shown here by immunoelectron microscopy to be located in axons. It is localized to straight filament variants of PHFs. The small amount of PHF-tau in white matter, therefore, appears not to result from the contamination of immunochemical preparations but to be an integral constituent of affected axons in AD. © 1994 Wiley-Liss, Inc.     

Human amyloid beta-induced neuroinflammation is an early event in neurodegeneration

Using a human amyloid beta (Abeta) intracerebroventricular infusion mouse model of Alzheimer's disease-related injury, we previously demonstrated that systemic administration of a glial activation inhibitor could suppress neuroinflammation, prevent synaptic damage, and attenuate hippocampal-dependent behavioral deficits. We report that Abeta-induced neuroinflammation is an early event associated with onset and progression of pathophysiology, can be suppressed by the glial inhibitor over a range of intervention start times, and is amenable to suppression without inhibiting peripheral tissue inflammatory responses. Specifically, hippocampal neuroinflammation and neurodegeneration occur in close time proximity at 4-6 weeks after the start of infusion. Intraperitoneal administration of inhibitor for 2-week intervals starting at various times after initiation of Abeta infusion suppresses progression of pathophysiology. The glial inhibitor is a selective suppressor of neuroinflammation, in that it does not block peripheral tissue production of proinflammatory cytokines or markers of B- and T-cell activation after a systemic lipopolysaccharide challenge. These results support a causal link between neuroinflammation and neurodegeneration, have important implications for future therapeutic development, and provide insight into the relative time window for targeting neuroinflammation with positive neurological outcomes.     

Neuron-selective toxicity of tau peptide in a cell culture model of neurodegenerative tauopathy: essential role for aggregation in neurotoxicity

Intracellular aggregation of tau is a pathological hallmark in Alzheimer's disease and other tauopathies. The mechanisms underlying tau aggregation and the role that these aggregates play in neuronal death have remained controversial. To study these issues, we established a cell culture model of tauopathy using a hexameric peptide with the sequence (306)VQIVYK(311) located within the third microtubule-binding repeat of tau, rendered cell-permeable by a tag of nine arginine residues (R(9)). This peptide (VQIVYK-R(9)), designated as T-peptide, self-assembles in vitro into paired helical filament-like aggregates. Primary neuronal cells treated with T-peptide die within 24 hr. Neurodegeneration correlates with the ability of the peptide to aggregate. Two peptides with mutations in the hexameric core, K-peptide (VQIVKK) and VV-peptide (VQVVVK), that are incapable of aggregating are not toxic, whereas two other mutant peptides, V-peptide (VQVVYK) and F-peptide (VQIVFK), which aggregate, are also neurotoxic. Two other peptides that aggregate in vitro, but are not derived from tau, are not neurotoxic suggesting sequence dependence. Although localizing to the nucleus, T-peptide induces aggregation of cellular proteins in the cytoplasm. These aggregates are not caused by disruption of endogenous tau localization, although endogenous tau is reduced in neurons exposed to T-peptide. Interestingly, nonneuronal cells are less sensitive to T-peptide toxicity, recapitulating in part the selective loss of neurons in tauopathies. Moreover, T-peptide treatment leads to mitochondrial dysfunction, a common feature of neurodegenerative disorders. The model system described here represents a convenient paradigm for studying the mechanisms underlying tau aggregation and neurotoxicity and for identifying compounds that can prevent these effects.     

Targeting microglia for the treatment of Alzheimer's Disease

While histological changes in microglia have long been recognized as a pathological feature of Alzheimer's disease (AD), recent genetic association studies have also strongly implicated microglia in the etiology of the disease. Coding and noncoding polymorphisms in several genes expressed in microglia—including APOE, TREM2, CD33, GRN, and IL1RAP—alter AD risk, and therefore could be considered as entry points for therapeutic intervention. Furthermore, microglia may have a substantial effect on current amyloid β (Aβ) and tau immunotherapy approaches, since they are the primary cell type in the brain to mediate Fc receptor‐facilitated antibody effector function. In this review, we discuss the considerations in selecting microglial therapeutic targets from the perspective of drug discovery feasibility, and consider the role of microglia in ongoing immunotherapy clinical strategies. GLIA 2016;64:1710–1732     

Neuropathology of familial tauopathy

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) is a hereditary progressive neurodegenerative disorder. FTDP‐17 was originally defined in Ann Arbor, Michigan, in 1996. Since then, more than 100 families with FTDP‐17 have been described throughout the world, including 18 families identified in Japan. Genetic studies have identified 40 different mutations in the microtubule‐associated protein tau (MAPT) gene. The clinical features of FTDP‐17 are characterized by behavioral, cognitive and motor disturbances that may occur in various combinations and degrees. Neuropathologic examination shows that various degrees of atrophy may be present in the frontal and temporal lobes, basal ganglia, amygdala and hippocampus. All the brains from patients with FTDP‐17 have also shown the presence of tau deposits in neurons and glial cells. Mutations in MAPT may result in the increased splicing of exon 10, leading to 4‐repeat tau depositions in both neurons and glial cells. MAPT mutations outside of exon 10 show 3‐ and 4‐repeat tau deposits, predominantly in neurons with less glial pathology. Neuronal pathology may resemble that of Alzheimer’s disease or Pick’s disease because of the presence of neurofibrillary tangles or Pick‐like bodies, whereas glial pathology may resemble that of progressive supranuclear palsy or corticobasal degeneration because of the presence of coiled bodies, tufted astrocytes or astrocytic plaques. Correlations between genetic mutations and the heterogeneity of clinical and neuropathologic features remain unclear.     

Data‐driven approaches for tau‐PET imaging biomarkers in Alzheimer's disease

Previous positron emission tomography (PET) studies have quantified filamentous tau pathology using regions‐of‐interest (ROIs) based on observations of the topographical distribution of neurofibrillary tangles in post‐mortem tissue. However, such approaches may not take full advantage of information contained in neuroimaging data. The present study employs an unsupervised data‐driven method to identify spatial patterns of tau‐PET distribution, and to compare these patterns to previously published “pathology‐driven” ROIs. Tau‐PET patterns were identified from a discovery sample comprised of 123 normal controls and patients with mild cognitive impairment or Alzheimer's disease (AD) dementia from the Swedish BioFINDER cohort, who underwent [¹⁸F]AV1451 PET scanning. Associations with cognition were tested in a separate sample of 90 individuals from ADNI. BioFINDER [¹⁸F]AV1451 images were entered into a robust voxelwise stable clustering algorithm, which resulted in five clusters. Mean [¹⁸F]AV1451 uptake in the data‐driven clusters, and in 35 previously published pathology‐driven ROIs, was extracted from ADNI [¹⁸F]AV1451 scans. We performed linear models comparing [¹⁸F]AV1451 signal across all 40 ROIs to tests of global cognition and episodic memory, adjusting for age, sex, and education. Two data‐driven ROIs consistently demonstrated the strongest or near‐strongest effect sizes across all cognitive tests. Inputting all regions plus demographics into a feature selection routine resulted in selection of two ROIs (one data‐driven, one pathology‐driven) and education, which together explained 28% of the variance of a global cognitive composite score. Our findings suggest that [¹⁸F]AV1451‐PET data naturally clusters into spatial patterns that are biologically meaningful and that may offer advantages as clinical tools.