Phosphorylated Tau Epitopes Research Articles

Granulovacuolar degeneration bodies are independently induced by tau and α-synuclein pathology

BackgroundGranulovacuolar degeneration bodies (GVBs) are intracellular vesicular structures that commonly accompany pathological tau accumulations in neurons of patients with tauopathies. Recently, we developed the first model for GVBs in primary neurons, that requires exogenous tau seeds to elicit tau aggregation. This model allowed the identification of GVBs as proteolytically active lysosomes induced by tau pathology. GVBs selectively accumulate cargo in a dense core, that shows differential and inconsistent immunopositivity for (phosphorylated) tau epitopes. Despite the strong evidence connecting GVBs to tau pathology, these structures have been reported in neurons without apparent pathology in brain tissue of tauopathy patients. Additionally, GVBs and putative GVBs have also been reported in the brain of patients with non-tau proteinopathies. Here, we investigated the connection between pathological protein assemblies and GVBs in more detail.MethodsThis study combined newly developed primary neuron models for tau and α-synuclein pathology with observations in human brain tissue from tauopathy and Parkinson’s disease patients. Immunolabeling and imaging techniques were employed for extensive characterisation of pathological proteins and GVBs. Quantitative data were obtained by high-content automated microscopy as well as single-cell analysis of confocal images.ResultsEmploying a novel seed-independent neuronal tau/GVB model, we show that in the context of tauopathy, GVBs are inseparably associated with the presence of cytosolic pathological tau and that intracellular tau aggregation precedes GVB formation, strengthening the causal relationship between pathological accumulation of tau and GVBs. We also report that GVBs are inseparably associated with pathological tau at the single-cell level in the hippocampus of tauopathy patients. Paradoxically, we demonstrate the presence of GVBs in the substantia nigra of Parkinson’s disease patients and in a primary neuron model for α-synuclein pathology. GVBs in this newly developed α-synuclein/GVB model are induced in the absence of cytosolic pathological tau accumulations. GVBs in the context of tau or α-synuclein pathology showed similar immunoreactivity for different phosphorylated tau epitopes. The phosphorylated tau immunoreactivity signature of GVBs is therefore independent of the presence of cytosolic tau pathology.ConclusionOur data identify the emergence of GVBs as a more generalised response to cytosolic protein pathology.

MultiTEP‐based conjugated vaccines targeting various phosphorylated tau epitopes are immunogenic and effective in the PS19 mouse model

AbstractBackgroundPathological tau correlates well with cognitive impairments in Alzheimer’s disease (AD) patients and represents a promising immunotherapy target. In theory, targeting an appropriate B‐cell epitope in pathological tau could effectively reduce the pathology without affecting normal native tau. Hyperphosphorylation of tau is a well‐known feature of Alzheimer’s and other tauopathies, with many disease‐specific phosphorylation sites documented. Accordingly, we attached several disease‐related phosphorylated tau peptides to our universal vaccine platform, MultiTEP that is safe and extremely immunogenic in various disease models of AD, rabbits, and aged monkeys. We hypothesized that high immunogenicity of MultiTEP‐based vaccines, coupled with the selectivity of the immune responses, will allow the identification of pathological and protective phosphorylation sites, thus leading to the generation of vaccines, which would preferentially clear the toxic forms of tau without disrupting the normal brain homeostasis.MethodA genetically engineered variant of MultiTEP was generated to enable chemoselective bioconjugation of synthetic peptides under mild aqueous conditions. MultiTEP‐based vaccines targeting p‐tau 202/205, p‐tau 396/404, p‐tau 422 were tested in the PS19 mouse model of AD and other tauopathies. Antibody specificity and titers were tested via ELISA. Brain total and phosphorylated soluble and insoluble tau were measured by ELISA and IHC.ResultAll three vaccines generated robust and selective immune responses specific to phosphorylated tau in mice. The generated antibodies selectively bound to the appropriate phosphorylated, but not non‐phosphorylated form of the same peptide. Importantly, these antibodies effectively reduce/cleared pathological tau in the brains of immunized mouse model of tauopathy. The MultiTEP immunogenicity was sufficient to generate robust immune responses after two immunizations, even without adjuvant.ConclusionMultiTEP‐based conjugated vaccines could be used to generate robust immune responses selective to multiple phosphorylation sites of tau. This, in conjunction with a previous report on MultiTEP‐based pE3Aβ vaccine, suggests that the methodology is universally applicable for various post‐translational modifications. Our technology can facilitate the identification of optimal therapeutic targets of p‐tau for generating an active immunotherapeutic strategy that can be used in conjunction with pE3Aβ vaccine. If successful, preventive vaccinations targeting disease‐related tau and/or Aβ molecules could at least delay AD and related neurodegenerative disorders.

Repopulated microglia induce expression of Cxcl13 with differential changes in Tau phosphorylation but do not impact amyloid pathology

BackgroundAdult microglia rely on self-renewal through division to repopulate and sustain their numbers. However, with aging, microglia display morphological and transcriptional changes that reflect a heightened state of neuroinflammation. This state threatens aging neurons and other cells and can influence the progression of Alzheimer’s disease (AD). In this study, we sought to determine whether renewing microglia through a forced partial depletion/repopulation method could attenuate AD pathology in the 3xTg and APP/PS1 mouse models.MethodsWe pharmacologically depleted the microglia of two cohorts of 21- to 22-month-old 3xTg mice and one cohort of 14-month-old APP/PS1 mice using PLX5622 formulated in chow for 2 weeks. Following depletion, we returned the mice to standard chow diet for 1 month to allow microglial repopulation. We assessed the effect of depletion and repopulation on AD pathology, microglial gene expression, and surface levels of homeostatic markers on microglia using immunohistochemistry, single-cell RNAseq and flow cytometry.ResultsAlthough we did not identify a significant impact of microglial repopulation on amyloid pathology in either of the AD models, we observed differential changes in phosphorylated-Tau epitopes after repopulation in the 3xTg mice. We provide evidence that repopulated microglia in the hippocampal formation exhibited changes in the levels of homeostatic microglial markers. Lastly, we identified novel subpopulations of microglia by performing single-cell RNAseq analysis on CD45int/+ cells from hippocampi of control and repopulated 3xTg mice. In particular, one subpopulation induced after repopulation is characterized by heightened expression of Cxcl13.ConclusionOverall, we found that depleting and repopulating microglia causes overexpression of microglial Cxcl13 with disparate effects on Tau and amyloid pathologies.

Tau phosphorylation sites serine202 and serine396 are differently altered in chronic traumatic encephalopathy and Alzheimer's disease.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with repetitive head impacts (RHI) typically sustained by contact sport athletes. Post-translation modifications to tau in CTE have not been well delineated or compared to Alzheimer's disease (AD). We measured phosphorylated tau epitopes within dorsolateral frontal cortex from post mortem brains with neither CTE nor AD (n = 108), CTE (n = 109), AD (n = 223), and both CTE and AD (n = 33). Levels of hyperphosphorylated tau (p-tau)202 , p-tau231 , and p-tau396 were significantly increased in CTE. Total years of RHI exposure was significantly associated with increased p-tau202 levels (P = .001), but not p-tau396 . Instead, p-tau396 was most closely related to amyloid beta (Aβ)1-42 levels (P < .001). The p-tau202 :p-tau396 ratio was significantly increased in early and late CTE compared to AD. In frontal cortex, p-tau202 is the most upregulated p-tau species in CTE, while p-tau396 is most increased in AD. p-tau202 and p-tau396 measurements may aid in developing biomarkers for disease.

Plasma p‐Tau181 and p‐Tau231 offer complementary information to identify Alzheimer's disease pathophysiology

AbstractBackgroundPathophysiological detection of Alzheimer’s disease (AD) is commonly made using cerebrospinal fluid (CSF) and positron emission tomography (PET). However, these methods are invasive and expensive, limiting their use in clinical settings Blood‐based biomarkers, mainly measuring phosphorylated tau epitopes at threonine 181 (p‐tau181), threonine 217 (p‐tau217), threonine 231 (p‐tau231), have shown promising results in detecting AD. However, it is still unknown whether the use of different plasma p‐Tau epitopes offer overlap or complementary information. Here, we tested the hypothesis that plasma p‐Tau181 and p‐Tau231 markers provide complementary information to each other to identify AD pathophysiology.MethodsPlasma p‐Tau181, p‐Tau231, [18F]AZD4694 amyloid‐PET, [18F]MK6240 tau‐PET, MRI and cognitive assessment from 284 individuals [30 cognitively unimpaired (CU) young, 155 CU), 54 mild cognitive impairment (MCI) and 38 AD] were obtained from the McGill TRIAD cohort. The individuals with diagnose of AD or MCI was classified as cognitively impaired (CI). Maximum Youden Index were used to determine cut‐offs for plasma p‐Tau181 (11.1 ng/mL) and plasma p‐Tau231 (11.8 ng/mL) considering CU young adults as controls. We classified the individual as positive or negative using plasma for each p‐Tau biomarker. Individuals were divided in the four possible groups (p‐Tau181‐/p‐Tau231‐, p‐Tau181+/p‐Tau231‐, p‐Tau181‐/p‐Tau231+ and p‐Tau181+/p‐Tau231+) and compared to assess differences in cognition and other biomarkers.ResultsPlasma p‐tau p‐Tau181 and p‐Tau231 showed moderate correlation with each other (r = 0.64, p &lt;0.0001) (Figure 1). The p‐Tau181+/p‐Tau231+ group presented higher tau PET and amyloid PET uptake compared to groups negative for both or only one p‐tau biomarker (all p&lt;0.01) (Figure 2A, B). Similarly, p‐Tau181+/p‐Tau231+ group showed worse cognitive performance and hippocampal atrophy than individuals with at least one p‐tau biomarker negative (Figure 2C‐E).ConclusionThese findings highlight ‐ for the first time ‐ that individuals positive for both p‐tau231 and p‐tau181 show worse cognitive performance, higher amyloid and tau burden, and lower hippocampus volume than individuals positive for only one of these markers. Our results have important implications for the use of plasma p‐tau biomarker in clinical trials and practice, suggesting that the quantification of more than one plasma p‐tau epitope may be useful to identify individuals on an AD pathway.

Internal Jugular Vein Compression Collar Mitigates Histopathological Alterations after Closed Head Rotational Head Impact in Swine: A Pilot Study

Recently, there has been increased concern about microstructural brain changes after head trauma. Clinical studies have investigated a neck collar that applies gentle bilateral jugular vein compression, designed to increase intracranial blood volume and brain stiffness during head trauma, which neuroimaging has shown to result in a reduction in brain microstructural alterations after a season of American football and soccer. Here, we utilized a swine model of mild traumatic brain injury to investigate the effects of internal jugular vein (IJV) compression on histopathological outcomes after injury. Animals were randomized to collar treatment (n = 8) or non-collar treatment (n = 6), anesthetized and suspended such that the head was supported by breakable tape. A custom-built device was used to impact the head, thus allowing the head to break the tape and rotate along the sagittal plane. Accelerometer data were collected for each group. Sham injured animals (n = 2) were exposed to anesthesia only. Following single head trauma, animals were euthanized and brains collected for histology. Whole slide immunohistochemistry was analyzed using Qupath software. There was no difference in linear or rotational acceleration between injured collar and non-collar animals (p > 0.05). Injured animals demonstrated higher levels of the phosphorylated tau epitope AT8 (p < 0.05) and the inflammatory microglial marker IBA1 (p < 0.05) across the entire brain, but the effect of injury was markedly reduced by collar treatment (p < 0.05) The current results indicate that internal jugular venous compression protects against histopathological alterations related to closed head trauma exposure.

HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology.

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. In Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 ± 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 ± 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P301L mutation hTau(P301L), and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-κB p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau(P301L), that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.

Immunotherapy Targeting Pathological Tau Conformers in a Tangle Mouse Model Reduces Brain Pathology with Associated Functional Improvements

Immunotherapies for various neurodegenerative diseases have recently emerged as a promising approach for clearing pathological protein conformers in these disorders. This type of treatment has not been assessed in models that develop neuronal tau aggregates as observed in frontotemporal dementia and Alzheimer's disease. Here, we present that active immunization with a phosphorylated tau epitope, in P301L tangle model mice, reduces aggregated tau in the brain and slows progression of the tangle-related behavioral phenotype. Females had more tau pathology than males but were also more receptive to the immunotherapy. The tau antibodies generated in these animals recognized pathological tau on brain sections. Performance on behavioral assays that require extensive motor coordination correlated with tau pathology in corresponding brain areas, and antibody levels against the immunogen correlated inversely with tau pathology. Interestingly, age-dependent autoantibodies that recognized recombinant tau protein but not the immunogen were detected in the P301L mice. To confirm that anti-tau antibodies could enter the brain and bind to pathological tau, FITC-tagged antibodies purified from a P301L mouse, with a high antibody titer against the immunogen, were injected into the carotid artery of P301L mice. These antibodies were subsequently detected within the brain and colocalized with PHF1 and MC1 antibodies that recognize pathological tau. Currently, no treatment is available for clearing tau aggregates. Our present findings may lead to a novel therapy targeting one of the major hallmarks of Alzheimer's disease and frontotemporal dementia.

Differential Changes in Phosphorylation of Tau at PHF-1 and 12E8 Epitopes During Brain Ischemia and Reperfusion in Gerbils

Cortical neurons are vulnerable to ischemic insult, which may cause cytoskeletal changes and neurodegeneration. Tau is a microtubule-associated protein expressed in neuronal and glial cells. We examined the phosphorylation status of tau protein in the gerbil brain cortex during 5 min ischemia induced by bilateral common carotid artery occlusion followed by reperfusion for 20 min to 7 days. Control brain homogenates contained 63, 65 and 68 kD polypeptides of tau immunoreactive with Alz 50, Tau 14 and Tau 46 antibodies raised against non-phosphorylated tau epitopes. Gerbil tau was also immunoreactive with some (PHF-1 and 12E8) but not all (AT8, AT100, AT180 and AT270) antibodies raised against phosphorylated tau epitopes. PHF-1 recognized a single 68 kD polypeptide and 12E8 bound the 63 kD polypeptide. During 5 min ischemia, PHF-1 immunoreactivity declined to 6%, then recovered to control levels after 20 min of blood recirculation and subsequently increased above control values 3 and 7 days later. In contrast, 12E8 immunoreactivity remained stable during ischemia and reperfusion. Our results suggest that the two phosphorylated epitopes of tau are regulated by different mechanisms and may play different roles in microtubule dynamics. They may also define various pools of neuronal/glial cells vulnerable to ischemia.

Measurement of Phosphorylated Tau Epitopes in the Differential Diagnosis of Alzheimer Disease: A Comparative Cerebrospinal Fluid Study

Measurement of Phosphorylated Tau Epitopes in the Differential Diagnosis of Alzheimer Disease: A Comparative Cerebrospinal Fluid Study

Transient Cerebral Ischemia Induces Aberrant Neuronal Cell Cycle Re-entry and Alzheimer's Disease-like Tauopathy in Female Rats

Aberrant mitosis occurs in many tauopathy-related neurodegenerative diseases and is believed to precede the formation of neurofibrillary tangles. In this study, we report for the first time that transient cerebral ischemia induces aberrant mitotic proteins and hyperphosphorylation of tau protein with neurofibrillary tangle-like conformational epitopes in adult female rat cortex. Following transient cerebral ischemia in rats, initiation of apoptosis precedes and is potentially integrated with subsequent aberrant mitosis and tau hyperphosphorylation. Furthermore, inhibition of mitosis-related cyclin-dependent kinases (Cdks) by roscovitine significantly reduced the hyperphosphorylation of tau. Administration of the female sex steroid and potent neuroprotective agent, 17beta-estradiol, reduced ischemia-reperfusion-induced cerebral damage and the subsequent aberrant mitosis and tauopathies. These results provide a neuropathological basis for the higher prevalence of dementia in stroke patients and support the hypothesis that apoptosis and aberrant mitosis are integrated pathological events in neurons that may play a critical role in the development of Alzheimer's disease and other tauopathy-related neuropathology.

Advances in the development of biomarkers for Alzheimer’s disease: from CSF total tau and Aβ 1–42 proteins to phosphorylated tau protein

Advances have been made to establish biological markers of Alzheimer’s disease (AD). Measurement of total tau (t-tau) and beta-amyloid 1–42 (Aβ 1–42) in the cerebrospinal fluid (CSF) seems useful to discriminate early and incipient AD from age-associated memory-impairment, depression, and some secondary dementias. New immunoassays to detect different phosphorylated tau epitopes (p-tau) have recently been developed. P-tau phosphorylated at threonine 231 (p-tau 231) showed improvements compared to t-tau in the early detection of AD in subjects with mild cognitive impairment. As p-tau 231 declined during the course of AD, it may have potential to track disease progression. Additionally, p-tau 231 improved differential diagnosis between AD, frontotemporal dementia, and geriatric major depression. P-tau phosphorylated at threonine 181 improved diagnostic accuracy between AD and dementia with Lewy bodies. P-tau phosphorylated at serine 199 demonstrated high discriminative power between AD and non-Alzheimer’s dementia. P-tau phosphorylated at serine 306/serine 404 improved differential diagnosis between AD and vascular dementia. A comparative study of the different p-tau epitopes is currently under way. In summary, first clinical multi-center studies suggest that measurement of phosphorylated tau proteins may significantly improve early and differential diagnosis and may come close to fulfilling proposed criteria of a biological marker for AD.

Iron-induced oxidative stress modify tau phosphorylation patterns in hippocampal cell cultures.

Oxidative stress phenomena have been related with the onset of neurodegenerative diseases. Particularly in Alzheimer Disease (AD), oxygen reactive species (ROS) and its derivatives can be found in brain samples of postmortem AD patients. However, the mechanisms by which oxygen reactive species can alter neuronal function are still not elucidated. There is a growing amount of evidence pointing to a role for mitochondrial damage as the source of free radicals involved in oxidative stress. Among the species that participate in the production of oxygen reactive radicals, transition metals are one of the most important. Several reports have implicated the involvement of redox-active metals with the onset of different neurodegenerative diseases such as Alzheimer's Disease (AD), Progressive Supranuclear Palsy (PSP), Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD). On the other hand, our previous studies have indicated that A beta-induced deregulation of the protein kinase Cdk5 associated with tau protein hyperphosphorylation constitute a critical pathway toward neurodegeneration. In the current paper we have shown that iron induces an imbalance in the function of Cdk5/p25 system of hippocampal neurons, resulting in a marked decrease in tau phosphorylation at the typical Alzheimer's epitopes. The loss of phosphorylated tau epitopes correlated with an increase in 4-hydroxy-nonenal (HNE) adducts revealing damage by oxidative stress. This effects on tau phosphorylation patterns seems to be a consequence of a decrease in the Cdk5/p25 complex activity that appears to result from a depletion of the activator p25, a mechanism in which calcium transients could be implicated.

Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease.

Microtubule associated protein tau is abnormally phosphorylated in Alzheimer's disease (AD) and aggregates as paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). We show here that the pattern of tau phosphorylation correlates with the loss of neuronal integrity. Studies using 11 phosphorylation dependent tau antibodies and a panel of AD cases of varying severity were evaluated in terms of three stages of neurofibrillary tangle development: (1) pre-neurofibrillary tangle, (2) intra-, and (3) extra-neuronal neurofibrillary tangles. The pretangle state, in which neurons display nonfibrillar, punctate regions in the cytoplasm, sound dendrites, somas, and nuclei, was observed especially with phospho-tau antibodies TG3 (pT231), pS262, and pT153. Intraneuronal neurofibrillary tangles are homogenously stained with fibrillar tau structures, which were most prominently stained with pT175/181, 12E8 (pS262/pS356), pS422, pS46, pS214 antibodies. Extracellular NFTs, which contain substantial filamentous tau, are most prominently stained with AT8 (pS199/pS202/pT205), AT100 (pT212/pS214), and PHF-1 (pS396/pS404) antibodies, which also stain intracellular NFT. The sequence of early tau phosphorylation suggests that there are events prior to filament formation that are specific to particular phosphorylated tau epitopes, leading to conformational changes and cytopathological alterations.

Site-specific Phosphorylation of Tau Accompanied by Activation of Mitogen-activated Protein Kinase (MAPK) in Brains of Niemann-Pick Type C Mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Developmental regulation and PKC dependence of Alzheimer's-type tau phosphorylations in cultured fetal rat hippocampal neurons

Attempts to describe a mechanism of neurofibrillary tangle formation often focus on site specific phosphorylations of tau protein. These have typically been described in both Alzheimer's disease and developing brains. Therefore, study of the developmental regulation of Alzheimer epitope tau phosphorylations may help explain their persistence or recurrence during Alzheimer's disease. Using fetal rat hippocampal cultures, we report a spatial and temporal expression of tau phosphorylation during neuronal differentiation. We have examined phosphorylation at the epitopes recognized by monoclonal antibodies, PHF-1 and Tau 1. Tau was highly phosphorylated at the PHF-1 epitope at all culture ages examined using both immunohistochemical staining and Western blots. Tau was heavily phosphorylated at the Tau 1 epitope only in older cultures. The populations of tau recognized by the two antibodies also exhibited different solubilities, suggesting different microtubule binding behaviors: tau phosphorylated at PHF-1 was retained in axons following solubilization whereas Tau 1 immunoreactive tau was not retained in any cell compartment. Finally, in this culture system, maintenance of phosphorylation at the PHF-1 epitope, but not the Tau 1 epitope, required protein kinase C activity. These results indicate unique regulatory mechanisms and roles for each of these phosphorylated tau epitopes.

Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.

Sporadic inclusion-body myositis (s-IBM) and the hereditary inclusion-body myopathies (h-IBMs) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers containing paired-helical filaments (PHFs). An interesting feature of the s- and h-IBM muscle phenotype is its striking similarity to Alzheimer-disease (AD) brain. We immunostained muscle biopsies of 9 s-IBM patients, 9 autosomal-recessive h-IBM patients, 1 autosomal-dominant h-IBM patients, and 18 normal and disease-controls with several antibodies known to react with the hyperphosphorylated tau of AD-PHFs. Those included SMI-31, SMI-310, PHF-1, and AT8. In both s- and h-IBM, virtually all vacuolated muscle fibers had strongly immunoreactive inclusions with SMI-31, and by immuno-electronmicroscopy SMI-31 was exclusively localized to PHFs. Approximately 40 to 50% of both s- and h-IBM vacuolated muscle fibers were also immunoreactive with AT8 antibody. To the contrary, in h-IBM, there was no immunoreactivity with SMI-310 and PHF-1 antibodies, whereas in s-IBM the vacuolated muscle fibers had strong immunoreactivity with those two antibodies. By immunoelectronmicorscopy, SMI-310 and PHF-1 also were localized to PHFs. Within s-IBM muscle fibers, the structures immunoreactive with SMI-310 were congophilic, whereas h-IBM muscle fibers did not have congophilia. Our studies: (a) demonstrate a distinct difference between s-IBM and the h-IBMs in regard to expression of immunoreactive phosphorylated tau and congophilia; (b) demonstrate a new "diagnostic duo" combination of SMI-31 and SMI-310 antibodies for identifying and distinguishing s-IBM and the h-IBMs; and (c) provide another close similarity of pathologic phenotypes between s-IBM muscle and AD brain, suggesting that similar cellular pathogenic mechanisms may be active in both diseases.

Phosphorylation of tau in apolipoprotein E-deficient mice

It has been suggested that the deleterious effects of the allele E4 of apolipoprotein E (apoE) in Alzheimer's disease (AD) are related to its inability to interact with the microtubule associated protein tau and to thereby prevent its hyperphosphorylation. In the present study we investigated the effects of apoE on tau phosphorylation by immunoblot analysis of the levels and extents of phosphorylation of tau of apoE-deficient mice. This revealed that mAb AT8, which is directed against a phosphorylated tau epitope, labels tau of the apoE-deficient mice more intensely than that of control mice and that the opposite occurs with mAb Taul, which is directed against dephosphorylated tau epitopes. mAb ALZ50 also labeled the tau enriched preparations of the apoE-deficient mice more intensely than those of the controls, whereas the extents of their labeling by the phosphorylation insensitive anti-tau mAb 134 were similar. These results suggest that tau of apoE-deficient mice is hyperphosphorylated.

Epitope expression and hyperphosphorylation of tau protein in corticobasal degeneration: differentiation from progressive supranuclear palsy.

Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial accumulation of abnormal tau protein. Using immunohistochemistry we analyzed tau epitope expression and phosphorylation state in CBD and compared them to cytoskeletal changes in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Epitopes spanning the entire length of the tau protein were present in CBD inclusions. An antibody against the alternatively spliced exon 3 did not recognize cytoskeletal lesions in CBD, but did in AD and PSP. Tau epitopes from each region of the molecule were present in cytoskeletal inclusions in CBD, including gray matter astrocytic plaques, gray and white matter threads, and oligodendroglial inclusions. As in AD, tau from CBD was highly phosphorylated. Antibodies that recognized phosphorylated tau epitopes reacted with material from CBD in a highly phosphatase-dependent manner. Again, all types of inclusions contained phosphorylated epitopes. We conclude that abnormal tau protein in CBD comprises the entire tau molecule and is highly phosphorylated, but is distinguished from AD and PSP by the paucity of epitopes contained in the alternatively spliced exon 3.

Microtubule-Associated Protein Tau Is Hyperphosphorylated during Mitosis in the Human Neuroblastoma Cell Line SH-SY5Y

A phosphorylated tau epitope specific for paired helical filaments in Alzheimer's disease is recognized by monoclonal antibody PHF-1. Healthy adult brains lack the PHF-1 epitope (PHF-1 tau), but it is transiently expressed by immature neurons during development. We have found that proliferating SH-SY5Y human neuroblastoma cells also express PHF-1 tau. Consistent with the recent finding that cell-cycle-dependent kinases can phosphorylate tau in vitro, flow cytometry showed that mitotic SH-SY5Y cells were up to 18-fold more PHF-1 immunoreactive than nonmitotic cells. On immunoblots, PHF-1 tau in mitotic and nonmitotic cells also was strikingly different. First, mitosis induced a prominent PHF-1 reactive band at 120 kDa, which likely accounted for the large increase in PHF-1 signal seen at mitosis. Although the size of the 120-kDa band is consistent with it being the high-molecular-weight form of tau, other antibodies to tau did not recognize it. Second, mitosis caused a hyperphosphorylation of the PHF-1 immunoreactive tau band normally seen at 50 kDa. In mitotic cells this band had an increased intensity and molecular weight. Alkaline phosphatase treatment abolished tau Mr heterogeneity, verifying that the variations in mobility were due to phosphorylation. These data show that cell-cycle-dependent hyperphosphorylation of tau occurs in intact cells, and they support the hypothesis that aberrant activity of cell-cycle-dependent kinases may contribute to tau phosphorylation and PHF formation in Alzheimer's disease.