Plasma tau protein in comatose patients after cardiac arrest treated with therapeutic hypothermia.

BACKGROUND: Neurological outcome after cardiac arrest (CA) is difficult to predict in the acute phase. In this pilot study, we assessed blood levels of tau protein as a prognostic marker for the neurological outcome after 6 months in patients treated with hypothermia after resuscitation from CA. METHODS: 22 unconscious patients resuscitated after CA were treated with mild hypothermia (32-34°C) for 26 h. Blood samples were collected at 2, 6, 12, 24, 48, and 96 h after CA, and the concentration of tau protein was analyzed. Neurological outcome was assessed with the Glasgow-Pittsburgh cerebral performance category (CPC) scale at intensive care unit (ICU) discharge and after 6 months. The higher of the two CPC scores was used. RESULTS: At ICU discharge, 21/22 patients were alive, of whom 10 had a good (CPC 1-2) outcome. After 6 months, 15/22 patients were alive, of whom 14 had a good outcome. Tau protein levels were higher among those with a poor outcome at 48 h and 96 h. At 96 h sampling, tau concentration predicted a poor outcome (CPC 3-5) with a sensitivity of 71% and a specificity of 93%. CONCLUSIONS: Although in a pilot study, a late increase in plasma tau protein seems to be associated with a worse outcome after hypothermia treatment after CA, although more studies are needed.

Evolution of Abeta42 and Abeta40 levels and Abeta42/Abeta40 ratio in plasma during progression of Alzheimer's disease: a multicenter assessment.

OBJECTIVE: To better understand the seemingly contradictory plasma beta-amyloid (Abeta) results in Alzheimer's disease (AD) patients by using a newly developed plasma Abeta assay, the INNO-BIA plasma Abeta forms, in a multicenter study. METHODS: A combined retrospective analysis of plasma Abeta isoforms on mild cognitive impairment (MCI) from three large cross-sectional studies involving 643 samples from the participating German and Swedish centers. RESULTS: Detection modules based on two different amino (N)-terminal specific Abeta monoclonal antibodies demonstrated that Abeta in plasma could be reliable quantified using a sandwich immunoassay technology with high precision, even for low Abeta42 plasma concentrations. Abeta40 and Abeta42 concentrations varied consistently with the ApoE genotype, while the Abeta42/Abeta40 ratio did not. Irrespective of the decrease of the Abeta42/Abeta40 ratio with age and MMSE, this parameter was strongly associated with AD, as defined in this study by elevated hyperphosphorylated (P-tau181P) levels in cerebrospinal fluid (CSF). CONCLUSION: A highly robust assay for repeatedly measuring Abeta forms in plasma such as INNO-BIA plasma Abeta forms might be a useful tool in a future risk assessment of AD.

Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies.

The primary feature of dementia with Lewy bodies (DLB) is the aggregation of alpha-synuclein into characteristic lesions: Lewy bodies (LBs) and Lewy neurites. However, in most of DLB cases, LBs are associated with neurofibrillary tangles and amyloid plaques (both Alzheimer disease [AD]-related lesions). We wanted to determine if this overlap of lesions is statistical, as a result of the late onset of both diseases, or results from a specific physiopathological synergy between synucleinopathy and either tauopathy or amyloid pathology. All patients with DLB from our prospective and multidisciplinary study were analyzed. These cases were compared with cases with pure AD and patients with Parkinson disease and controls. All cases were analyzed thoroughly at the neuropathologic and biochemical levels with a biochemical staging of aggregated alpha-synuclein, tau, and Abeta species. All sporadic cases of DLB were associated with abundant deposits of Abeta x-42 that were similar in quality and quantity to those of AD. Amyloid precursor protein (APP) dysfunction is a risk factor for AD as demonstrated by pathogenic mutations and Abeta accumulation. The constant and abundant Abeta x-42 deposition in sporadic DLB suggests that synucleinopathy is also promoted by APP dysfunction. Therefore, we conclude that APP is a therapeutic target for both AD and DLB.

Disease- and treatment-related elevation of the neurodegenerative marker tau in children with hematological malignancies.

Children acquire neuropsychologic dysfunctions after chemotherapy for hematologic malignancy. In this study, putative changes in levels of CSF-tau (a marker of neural dysintegrity) in leukemic children prior to and during chemotherapy were studied. Cerebrospinal fluid (CSF) samples were obtained before and during treatment from patients with B cell non-Hodgkin's lymphoma (NHL, n = 10), non-B cell acute lymphoblastic leukemia/NHL (non-B-ALL, n = 48), acute myeloid leukemia (AML, n = 9), other malignant diseases (n = 9), and six control children. A sandwich-type ELISA (INNOTEST hTAU-Ag) was used for measuring CSF-tau. Sixteen out of 50 patients with hematological malignancies, including the patients with proven leukemic CNS invasion, already showed high CSF-tau levels at baseline (>300 pg/ml). The pre-induction treatment for non-B-ALL, consisting of only corticosteroids and methotrexate (MTX), resulted in a significant increase of tau at day 8 (on average to 535 pg/ml). Larger increases as compared to baseline levels of CSF-tau were observed in patients treated for B-NHL with systemic vincristine, corticosteroids and cyclophosphamide, and intrathecal MTX (mean 776 pg/ml at day 8). In two AML patients with CNS invasion, CSF-tau increased during chemotherapy up to 1,500 and 948 pg/ml, respectively. In one non-B-ALL patient with MTX-induced clinical neurotoxicity, CSF-tau was above the detection limit of 2,000 pg/ml. Almost one-third of the patients with hematological malignancies had elevated CSF-tau levels at diagnosis. Transient high levels of CSF-tau, reaching levels observed in other neurodegenerative disorders, were observed during induction chemotherapy for non-B-ALL, B-NHL and CNS+ AML. The clinical implications of both observations will be the subject of further study.

Postmortem changes in the phosphorylation state of tau-protein in the rat brain.

The phosphorylation state of tau-protein is crucial for the regulation of neuronal microtubule organization. Functional conclusions on tau-protein require an accurate assessment of phosphorylated sites. Therefore, the in vivo distribution and postmortem preservation of some phospho-epitopes on tau-protein were examined in the rat brain under different fixation and preparation conditions. Detection of tau-protein with a phosphorylation-independent antiserum revealed both axonal and somatodendritic localizations, which were not influenced by a postmortem interval of 30 min. The phospho-epitopes recognized by 12E8, AT8, and PHF-1 were mainly localized in the somatodendritic compartment. The binding sites of AT8 and PHF-1 were rapidly dephosphorylated postmortem, whereas the Tau-1 epitope was unmasked in the somatodendritic region. The axonally located phospho-epitope of AT270 and the nuclear epitope of AT100 were still detectable after a postmortem interval of 30 min. Postmortem dephosphorylation and inhibition of this process by PP1 and/or PP2A was further demonstrated on Western blot. In conclusion, rapid processing of tau-protein is essential for the correct assessment of investigations on phospho-isoforms.

Association of CSF apolipoprotein E, Abeta42 and cognition in Alzheimer's disease.

A significant association between CSF Abeta42 and cognition in patients with Alzheimer's disease (AD) homozygous for the epsilon3 allele of the apolipoprotein E (apoE) has been described. In this study we extended our observations on apoE, as another plaque component, and investigated the association between CSF apoE concentrations and cognitive performance after stratification for the apoE genotype in 62 patients with AD, 19 other forms of dementia and 18 controls. CSF Abeta42 and apoE concentrations were significantly and positively associated with Mini Mental State Examination (MMSE) score in AD (Abeta42: r = 0.332; P = 0.026; apoE: r = 0.386; P = 0.006). For Abeta42 this association was exclusively present in epsilon3 homozygotes (r = 0.44; P = 0.014), whereas apoE was correlated with MMSE in epsilon4 hetero- or homozygotes subjects (epsilon4/epsilonX: r = 0.638; P = 0.004: epsilon4/epsilon4; r = 0.812; P = 0.05). No association was observed between CSF concentrations of Abeta42 and apoE. The significant relationship between MMSE and CSF Abeta42 in epsilon3 homozygotes and apoE in epsilon4 hetero- and homozygotes respectively may suggest that both proteins may be associated independently from each other with cognitive decline.

Molecular dissection of the paired helical filament.

Abundant neurofibrillary tangles, neuropil threads and plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease where their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in a hyperphosphorylated state. PHF-tau is hyperphosphorylated on six adult brain tau isoforms. As a consequence, tau is unable to bind to microtubules and is believed to self-assemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues play an important role in the hyperphosphorylation of tau. Candidate protein kinases include mitogen-activated protein kinase, glycogen synthase kinase-3 and cyclin-dependent kinase 5, whereas the trimeric form of protein phosphatase 2A is a candidate phosphatase.

CSF total tau, Abeta42 and phosphorylated tau protein as biomarkers for Alzheimer's disease.

With the arrival of effective symptomatic treatments and the promise of drugs that may delay progression, we now need to identify Alzheimer's disease (AD) at an early stage of the disease. To diagnose AD earlier and more accurately, attention has been directed toward peripheral biochemical markers. This article reviews promising potential cerebrospinal fluid (CSF) biomarkers for AD focussing on their role in clinical diagnosis. In particular, two biochemical markers, CSF total tau (t-tau) protein and the 42 amino acid form of beta-amyloid (Abeta42), perform satisfactorily enough to achieve a role in the clinical diagnostic settings of patients with dementia together with the cumulative information from basic clinical work-up, genetic screening, and brain imaging. These CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. In order to discriminate AD from other primary dementia disorders, however, more accurate and specific markers are needed. Preliminary evidence strongly suggests that quantification of tau phosphorylated at specific sites in CSF improves early detection, differential diagnosis, and tracking of disease progression in AD.

Evaluation of CSF-tau and CSF-Abeta42 as diagnostic markers for Alzheimer disease in clinical practice.

OBJECTIVE: To evaluate the diagnostic potential of cerebrospinal fluid (CSF) levels of tau and beta-amyloid protein ending at amino acid 42 (Abeta42) as biomarkers for Alzheimer disease (AD) in clinical practice. DESIGN: A 1-year prospective study. SETTING: Community population-based sample of all consecutive patients admitted for investigation of cognitive symptoms to the Piteå River Valley Hospital, Piteå, Sweden. PATIENTS: A total of 241 patients with probable AD (n = 105), possible AD (n = 58), vascular dementia (n = 23), mild cognitive impairment (n = 20), Lewy body dementia (n = 9), other neurological disorders (n = 3), and psychiatric disorders (n = 5) and nondemented individuals (n = 18). MAIN OUTCOME MEASURES: Cerebrospinal fluid tau and CSF-Abeta42 were assayed each week as routine clinical neurochemical analyses. Sensitivity and specificity were defined using the regression line from 100 control subjects from a multicenter study. Positive and negative predictive values were calculated for different prevalence rates of AD. RESULTS: We found increased CSF-tau and decreased CSF-Abeta42 levels in probable and possible AD. Sensitivity was 94% for probable AD, 88% for possible AD, and 75% for mild cognitive impairment, whereas specificity was 100% for psychiatric disorders and 89% for nondemented. Specificity was lower in Lewy body dementia (67%) mainly because of low CSF-Abeta42 levels and in vascular dementia (48%) mainly because of high CSF-tau levels. Sensitivity for CSF-tau and CSF-Abeta42 increased in patients with AD possessing the ApoE epsilon4 allele, approaching 100%. At a prevalence of AD of 45%, the positive predictive value was 90% and the negative predictive value was 95%. CONCLUSIONS: Cerebrospinal fluid tau and CSF-Abeta42 have so far been studied in research settings, under conditions providing data on the optimal performance. We examined a prospective patient sample, with assays run in clinical routine, giving figures closer to the true performance of CSF-tau and CSF-Abeta42. The predictive value for AD was greater than 90%. Therefore, these biomarkers may have a role in the clinical workup of patients with cognitive impairment, especially to differentiate early AD from normal aging and psychiatric disorders.

Cerebrospinal fluid beta-amyloid(1-42) in Alzheimer disease: differences between early- and late-onset Alzheimer disease and stability during the course of disease.

OBJECTIVES: To study the diagnostic potential of the 42 amino acid form of beta-amyloid (beta-amyloid(1-42)) in cerebrospinal fluid (CSF) as a biochemical marker for Alzheimer disease (AD), the intra-individual biological variation of CSF-beta-amyloid(1-42) level in patients with AD, and the possible effects of differential binding between beta-amyloid and apolipoprotein E isoforms on CSF-beta-amyloid(1-42) levels. DESIGN: A 20-month prospective follow-up study. SETTING: Community population-based sample of consecutive patients with AD referred to the Piteå River Valley Hospital, Piteå, Sweden. PATIENTS: Fifty-three patients with AD (mean +/- SD age, 71.4 +/- 7.4 years) diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association criteria and 21 healthy, age-matched (mean +/- SD age, 68.8 +/- 8.0 years) control subjects. MAIN OUTCOME MEASURES: Cerebrospinal fluid beta-amyloid(1-42) level--analyzed using enzyme-linked immunosorbent assay--and severity of dementia--analyzed using the Mini-Mental State Examination. RESULTS: Mean +/- SD levels of CSF-beta-amyloid(1-42) were decreased (P<.001) in patients with AD (709 +/- 304 pg/mL) compared with controls (1678 +/- 436 pg/mL). Most patients with AD (49 [92%] of 53 patients) had reduced levels (<1130 pg/mL). A highly significant correlation (r = 0.90; P<.001) between baseline and 1-year follow-up CSF-beta-amyloid(1-42) levels was found. There were no significant correlations between CSF-beta-amyloid(1-42) level and duration (r = -0.16) or severity (r = -0.02) of dementia. Low levels were also found in patients with mild dementia (Mini-Mental State Examination score, >25). CONCLUSIONS: The sensitivity of CSF-beta-amyloid(1-42) level as a diagnostic marker for AD is high. The intra-individual biological variation in CSF-beta-amyloid(1-42) level is low. Low CSF-beta-amyloid(1-42) levels are also found in the earlier stages of dementia in patients with AD. These findings suggest that CSF-beta-amyloid(1-42) analyses may be of value in the clinical diagnosis of AD, especially in the early course of the disease, when drug therapy may have the greatest potential of being effective but clinical diagnosis is particularly difficult.

Cerebrospinal fluid tau and beta-amyloid(1-42) in dementia disorders.

The reliability of cerebrospinal fluid (CSF)-tau and CSF-beta-amyloid assays for diagnosis of Alzheimer's disease and other dementing disorders such as frontotemporal dementia (FTD), dementia with Lewy bodies (DLB) and Creutzfeldt-Jakob disease (CJD) is reviewed. CSF assessment of the two proteins is useful in early diagnosis of AD and to differentiate it from FTD and DLB. Extremely high CSF-tau levels can discriminate CJD from AD.

Sensitivity, specificity, and stability of CSF-tau in AD in a community-based patient sample.

OBJECTIVE: To evaluate the sensitivity and specificity of CSF-tau in clinical practice as a diagnostic marker for AD compared with normal aging and depression, to study the stability of CSF-tau in longitudinal samples, and to determine whether CSF-tau levels are influenced by different covariates such as gender, age, duration or severity of disease, or possession of the APOE-epsilon4 allele. METHODS: Consecutive AD patients from a community-based sample were studied, including 407 patients with AD (274 with probable AD and 133 with possible AD), 28 patients with depression, and 65 healthy elderly control subjects. A follow-up lumbar puncture was performed in 192 AD patients after approximately 1 year. CSF-tau was determined using a sandwich ELISA, which was run as a routine clinical neurochemical analysis. RESULTS: CSF-tau was increased in probable (690+/-341 pg/mL; p < 0.0001) and possible (661+/-447 pg/mL; p < 0.0001) AD, but not in depression (231+/-110 pg/mL) compared with control subjects (227+/-101 pg/mL). Receiver operating characteristics analysis showed that a cutoff level of 302 pg/mL resulted in a sensitivity of 93% (95% CI, 90-96%) and a specificity of 86% (95% CI, 75-94%), with an area under the curve of 0.95 to discriminate AD from control subjects. Within the AD group, CSF-tau did not differ significantly between baseline and follow-up investigations, and was relatively stable between baseline and 1-year follow-up levels, with a coefficient of variation of 21.0%. High CSF-tau levels were also found in most AD patients with very short duration of dementia, and with Mini-Mental State Examination scores >23 (n = 205). In total, 193 of 205 patients (sensitivity, 94%) had a CSF-tau level higher than 302 pg/mL. CONCLUSIONS: CSF-tau has a high sensitivity and specificity to differentiate AD from normal aging and depression, as demonstrated in a large community-based series of consecutive AD patients during which analyses were run continually in a clinical neurochemical laboratory. The increase in CSF-tau is found very early in the disease process in AD, is stable over time, and has a low interindividual variation on repeated sampling. Although high CSF-tau is found in some neurologic conditions (e.g., stroke), these findings suggest that CSF-tau may be of use to help in differentiating AD from normal aging and depression, especially early in the course of the disease, when the symptoms are vague and the diagnosis is especially difficult.

Improved discrimination of AD patients using beta-amyloid(1-42) and tau levels in CSF.

OBJECTIVE: To evaluate CSF levels of beta-amyloid(1-42) (Abeta42) alone and in combination with CSF tau for distinguishing AD from other conditions. METHODS: At 10 centers in Europe and the United States, 150 CSF samples from AD patients were analyzed and compared with 100 CSF samples from healthy volunteers or patients with disorders not associated with pathologic conditions of the brain (CON), 84 patients with other neurologic disorders (ND), and 79 patients with non-Alzheimer types of dementia (NAD). Sandwich ELISA techniques were used on site for measuring Abeta42 and tau. RESULTS: Median levels of Abeta42 in CSF were significantly lower in AD (487 pg/mL) than in CON (849 pg/mL; p = 0.001), ND (643 pg/mL; p = 0.001), and NAD (603 pg/mL; p = 0.001). Discrimination of AD from CON and ND was significantly improved by the combined assessment of Abeta42 and tau. At 85% sensitivity, specificity of the combined test was 86% (95% CI: 81% to 91%) compared with 55% (95% CI: 47% to 62%) for Abeta42 alone and 65% (95% CI: 58% to 72%) for tau. The combined test at 85% sensitivity was 58% (95% CI: 47% to 69%) specific for NAD. The APOE e4 gene load was negatively correlated with Abeta42 levels not only in AD but also in NAD. CONCLUSIONS: The combined measure of CSF Abeta42 and tau meets the requirements for clinical use in discriminating AD from normal aging and specific neurologic disorders.

Generation and characterization of mouse microglial cell lines.

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific esterase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histiocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-alpha, IL-1, IL-6, TGF-beta, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-alpha, IL-1, and TGF-beta. Our newly established murine microglial lines may prove to be useful models to study inflammation and repair in the brain.

Cerebrospinal fluid markers for Alzheimer's disease evaluated after acute ischemic stroke.

Potential cerebrospinal fluid (CSF) markers for Alzheimer's disease (AD) include tau protein, the 42 amino-acid form of amyloid beta (amyloid beta(1-42)) and apolipoprotein E (apoE). To study new aspects of these protein markers, we examined consecutive CSF samples from 26 patients with acute ischemic stroke. CSF samples were taken on day 0-1, day 2-3, day 7-9, 3 weeks and 3-5 months after the stroke. CSF-tau showed a marked increase day 2-3, which peaked after 1 week and returned to normal after 3-5 months. CSF-tau also showed correlation (r=0.95; p<0.01) with the size of the infarct. In contrast, CSF-amyloid beta(1-42) and CSF-apoE showed no significant changes during the period. The marked increase in CSF-tau levels after acute ischemic stroke indicate that CSF-tau reflect the degree of neuronal damage. The reason for unchanged levels of CSF-amyloid beta(1-42) and CSF-apoE after ischemic stroke remains unclear.

Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluid and plasma.

The standardization and clinical validation of the measurement of beta-amyloid(1-42) (Abeta42) in cerebrospinal fluid (CSF), plasma and urine is described using a commercially available sandwich-type ELISA with 21F12 and 3D6 as monoclonal antibodies. The INNOTEST beta-amyloid(1-42) allows the specific and reliable measurement of(1-42) amyloid peptides in CSF and plasma. The Abeta42 concentrations in serum and urine were below the detection limit. In plasma, no differences were found in Abeta42 levels between controls and patients with different neurodegenerative disorders (Alzheimer's disease (AD), Lewy body disease (LBD), others). In contrast, CSF-Abeta42 concentrations were lower in AD and LBD patients as compared to controls. No correlation was found in AD patients between CSF and plasma concentrations of Abeta42 or between CSF Abeta42 levels and blood-brain-barrier function. The quantitative outcome of the test is in part dependent on confounding factors such as tube type, freeze/thaw cycles, temperature of incubation, standard preparation protocol, and antibody selection. Notwithstanding these aspects, it emerged that Abeta42 is a useful biochemical marker for the diagnosis of AD patients, but there is a need for an international Abeta standard, a universally accepted protocol for CSF preparation, and a thorough evaluation of assay performance in function of the boundary conditions.

An in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric oxide and tumor necrosis factor-alpha.

The precise function of activated microglia and their secretory products remains controversial. In order to assess the role of microglial secretion products, we established an in vitro model of an inflammatory reaction in the brain by co-culturing microglial and neuronal cell lines. Upon stimulation with interferon-gamma and lipopolysaccharides, the microglial cells adopted an activated phenotype and secreted tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) and nitric oxide (NO). Neuronal degeneration was quantified by measuring the concentrations of microtubule associated protein tau and neuron specific enolase, which are also used as diagnostic tool in Alzheimer's disease, in supernatants. In activated contact co-cultures, the levels of these neuronal markers were significantly raised compared to non-activated co-cultures. NO-synthase inhibitors significantly diminished the rise of tau in activated co-cultures, while indomethacin, superoxide dismutase, or a neutralizing TNF-alpha antibody did not. When a chemical NO-donor or TNF-alpha were added to pure neuronal cultures, cell viability was significantly reduced. TNF-alpha increased neuronal sensitivity towards NO. There were indications that a part of the cells died by apoptosis. This model demonstrates a neurotoxic role for NO in microglia-induced neurodegeneration and provides a valuable in vitro tool for the study of microglia-neuron interactions during inflammation in the brain.

Expression of a paired helical filament tau epitope in embryonic chicken central nervous system.

Immunoblots from embryonic chicken optic lobes (midbrain) and spinal cord were found to contain different proteins with a molecular weight between 45 and 70 kDa that are recognized by monoclonal antibodies (mAbs) directed at human tau proteins. Each appears as a doublet on SDS-PAGE. The upper, slower migrating bands were recognized by AT8, a monoclonal antibody that requires a phosphorylated Ser-202, an epitope specific for abnormally phosphorylated tau of paired helical filaments (PHF-tau) in Alzheimer brains. The corresponding faster migrating bands were stained by the mAbs BT2 and tau-1, both requiring an epitope containing a non-phosphorylated Ser-202. Phosphatase treatment abolished binding of AT8 and induced an additional binding of tau-1 (and BT2) to the upper bands. Thus, the data suggest that in embryonic chicken central nervous system Ser-202 occurs in a phosphorylated and in a non-phosphorylated state in several distinct tau isoforms.

Developmental expression of tau proteins in the chicken and rat brain: rapid down-regulation of a paired helical filament epitope in the rat cerebral cortex coincides with the transition from immature to adult tau isoforms.

The monoclonal antibodies TAU-1 and AT8 are directed at human microtubule-associated protein tau epitopes that contain a dephosphorylated and phosphorylated Ser202, respectively, while AT180 and AT270 are anti-tau monoclonals with epitopes that require phosphorylated Thr181 and Thr231, respectively. We used these antibodies to study the developmental profiles of tau proteins in rat cerebral cortex and chicken optic lobes. In tau extracts from perinatal rat cerebral cortex. AT8 recognized one major protein band of approximately 50 kDa that peaks on postnatal day 6 and declines rapidly to lower levels at day 12. At later stages, the AT8 epitope was expressed by several adult tau isoforms that were, however, stained only very faintly in highly enriched samples. Two additional tau epitopes recognized by AT180 and AT270 were found to be expressed by one or two protein bands up to about postnatal day 19 and then declined. Unlike the AT8 epitope, in the mature brain these epitopes were stained strongly in enriched samples, where they were expressed by a greater number of adult isoforms. Between embryonic day 19 and postnatal day 12, TAU-1 was found to recognize one major protein band of approximately 50 kDa which migrated slightly faster than the AT8-binding band. At postnatal day 19 and all older stages (including adult cortex), at least three additional TAU-1-binding isoforms with higher apparent molecular weights were present. Hence, the transition from one immature to several adult TAU-1-binding tau isoforms between postnatal day 12 and 19 in rat cerebral cortex coincides with the phase of rapid down-regulation of the AT8 epitope. As in the rat cerebrum, in chicken optic lobes there is a developmental decrease of AT8-binding proteins which is paralleled by striking changes in the electrophoretic pattern of tau isoforms recognized by TAU-1. In both rat cerebral cortex and chicken optic lobes, the period of maximal expression of AT8-binding tau is morphologically characterized by intense axonal growth and beginning synaptogenesis, whereas its subsequent rapid down-regulation and the appearance of novel TAU-1-binding isoforms correlates with synaptic maturation, the onset of spontaneous electrical activity and the beginning of myelination.

Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205.

Hyperphosphorylated microtubule-associated protein tau is the major component of the paired helical filament of Alzheimer's disease. Phosphorylation-dependent anti-tau antibodies are being used to identify specific amino acids that are phosphorylated in tau from normal brain and Alzheimer's disease brain. As such, monoclonal antibody AT8 is widely used. By a combination of site-directed mutagenesis of recombinant tau and in vitro phosphorylation, we show that AT8 requires tau protein to be phosphorylated at both serine 202 and threonine 205 (using the numbering of the longest human brain tau isoform.