Elevated Tau Level in Aged Rat Cerebrospinal Fluid Reduced by Treatment with a Neurotrophic Compound.

Alzheimer's disease (AD) is the single major cause of dementia in middle- to old-age individuals, and, as of yet, no disease-modifying therapeutic drug is available for its treatment. A major obstacle in the successful development of disease-modifying therapeutic drugs has been the lack of suitable animal models of the sporadic form of AD as well as a biomarker that can be used both for therapeutic preclinical studies and for human clinical trials. Previously we showed neurogenesis and neuronal plasticity deficits and cognitive impairment and their rescue with a neurotrophic peptidergic compound, DGGLAG named P021, in aged Fisher rats. Here we show that P021 is blood-brain-barrier-permeable, and chronic oral treatment with this compound can reduce the brain level of total tau in the aged rats. Furthermore, cerebrospinal fluid (CSF) levels of both tau and Aß/AßPP are elevated in the aged animals, and chronic treatment with P021 can reduce tau but not Aß/AßPP to that of the levels found in young adult rats. Importantly, P021 does not induce any detectable immune reaction in rats. Collectively, these studies show the therapeutic potential of P021 as a disease-modifying compound and the suitability of the aged Fisher rats as a model of cerebral aging in which the therapeutic efficacy of a tau-reducing compound can be monitored in the CSF.

Intracerebroventricular streptozotocin exacerbates Alzheimer-like changes of 3xTg-AD mice.

Alzheimer's disease (AD) involves several possible molecular mechanisms, including impaired brain insulin signaling and glucose metabolism. To investigate the role of metabolic insults in AD, we injected streptozotocin (STZ), a diabetogenic compound if used in the periphery, into the lateral ventricle of the 6-month-old 3xTg-AD mice and studied the cognitive function as well as AD-like brain abnormalities, such as tau phosphorylation and Aß accumulation, 3-6 weeks later. We found that STZ exacerbated impairment of short-term and spatial reference memory in 3xTg-AD mice. We also observed an increase in tau hyperphosphorylation and neuroinflammation, a disturbance of brain insulin signaling, and a decrease in synaptic plasticity and amyloid ß peptides in the brain after STZ treatment. The expression of 20 AD-related genes, including those involved in the processing of amyloid precursor protein, cytoskeleton, glucose metabolism, insulin signaling, synaptic function, protein kinases, and apoptosis, was altered, suggesting that STZ disturbs multiple metabolic and cell signaling pathways in the brain. These findings provide experimental evidence of the role of metabolic insult in AD.

CSF biomarker variability in the Alzheimer's Association quality control program.

BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid beta 1-42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories. METHODS: Data from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Mölndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection. RESULTS: A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1-42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP). CONCLUSIONS: The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.

Cytochrome P450-7B1 and 11ß-hydroxysteroid dehydrogenase type 1 distribution in human tissues.

Abstract Successive action of cytochrome P450-7B1 (CYP7B1) and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) on 3ß-hydroxysteroids such as DHEA and epiandrosterone leads to the production of cytoprotective 7ß-hydroxylated derivatives. Investigation of the presence of these enzymes in human tissues could be carried out on commercially available human tissue arrays with use of antibodies specific to CYP7B1 and 11ß-HSD1 for immunohistochemistry. Both enzymes were detected mainly in tissues of endodermic and ectodermic origin which are prone to undergo inflammation. As low doses of the 7ß-hydroxylated derivatives of DHEA and epiandrosterone trigger the resolution of inflammation and tissue repair, CYP7B1 and 11ß-HSD1 tissue contents may reflect the tissue ability for reparation after pathological conditions.

The Alzheimer's Association external quality control program for cerebrospinal fluid biomarkers.

BACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid ß (Aß)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program. METHODS: The program is open for laboratories using commercially available kits for Aß, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Mölndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples. RESULTS: Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aß-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aß triplex (AßN-42, AßN-40, and AßN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories. CONCLUSIONS: Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.

Blood-cerebrospinal fluid barrier permeability in Alzheimer's disease.

The role of blood-cerebrospinal fluid barrier (BCB) dysfunction in Alzheimer's disease (AD) has been addressed but not yet established. We evaluated the BCB integrity in 179 samples of cerebrospinal fluid (CSF) retrospectively collected from AD patients and control cases using both CSF/serum albumin ratio (QAlb) and CSF secretory Ca2+-dependent phospholipase A2 (sPLA2) activity. These analyses were supplemented with the measurement of total tau, amyloid-ß1-42 (Aß1-42), and ubiquitin CSF levels. We found that due to its higher sensitivity, CSF sPLA2 activity could 1) discriminate AD from healthy controls and 2) showed BCB impairment in neurological control cases while QAlb could not. Moreover, the CSF sPLA2 activity measurement showed that around half of the AD patients were characterized by a BCB impairment. The BCB dysfunction observed in AD was independent from Mini-Mental State Examination score as well as CSF levels of total tau, Aß1-42, and ubiquitin. Finally, the BCB dysfunction was not limited to any of the CSF biomarkers-based previously identified subgroups of AD. These results suggest that the BCB damage occurs independent of and probably precedes both Aß and tau pathologies in a restricted subgroup of AD patients.

Cerebrospinal fluid secretory Ca2+-dependent phospholipase A2 activity: a biomarker of blood-cerebrospinal fluid barrier permeability.

The blood-brain barrier, the blood-cerebrospinal fluid barrier (BCB) and other specialized brain barriers are increasingly recognized as a major obstacle to the treatment of most brain disorders. The impairment of these barriers has been implicated in neuropathology of several diseases, such as autism, ischemia, multiple sclerosis and Alzheimer disease. This dual function of the blood-neural barriers points out the importance and need for the development of techniques that can evaluate the nature and level of their integrity. Here we report the discovery of CSF secretory Ca(2+)-dependent phospholipase A2 (sPLA2) activity as a measure of BCB permeability. Lumbar CSF from BCB impaired (n=26), multiple sclerosis (n=18) and healthy control (n=32) cases was analyzed using both a newly developed continuous fluorescence assay for CSF sPLA2 activity and CSF/Serum albumin ratio (Q(Alb)), the most common and established method to evaluate BCB permeability. While both measurements showed no significant differences between multiple sclerosis and age-matched normal healthy cases, they were highly correlated. Though the CSF sPLA2 activity and Q(Alb) had over 95% agreement, the former was found to be more sensitive than the latter in measuring low levels of BCB impairment.

Cerebrospinal fluid secretory Ca2+-dependent phospholipase A2 activity is increased in Alzheimer disease.

BACKGROUND: The phospholipase A(2) (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). METHODS: We used liposomes composed of a fluorescent probe (bis-Bodipy FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-alpha-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. RESULTS: Using 5 microL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca(2+)-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 micromol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20-77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. CONCLUSIONS: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

Dehydroepiandrosterone 7alpha-hydroxylation in human tissues: possible interference with type 1 11beta-hydroxysteroid dehydrogenase-mediated processes.

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7alpha-hydroxy-DHEA that is a substrate for 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which exists in the same tissues and carries out the inter-conversion of 7alpha- and 7beta-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11beta-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7alpha-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11beta-HSD1. Human skin was selected then and used to test its ability to produce 7alpha-hydroxy-DHEA, and to test the interference of 7alpha- and 7beta-hydroxy-DHEA and 7-oxo-DHEA with the 11beta-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11beta-HSD1 in the liver, skin and tonsils. DHEA was readily 7alpha-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11beta-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA was competitive with a Ki at 1.85+/-0.495 and 0.255+/-0.005 microM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a Ki at 1.13+/-0.15 microM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7alpha-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.

Dehydroepiandrosterone metabolites and their interactions in humans.

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochrome P4507B1 in the liver, skin and brain, which are targets for glucocorticoids. 7alpha-Hydroxy-DHEA produced anti-glucocorticoid effects in vivo but the interference between the glucocorticoid hormone binding with its receptor could not be determined. In the organs mentioned above, circulating inactive cortisone is reduced to active cortisol by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 7alpha-Hydroxy-DHEA is also a substrate for this enzyme. Studies of 11beta-HSD1 action on 7alpha-hydroxy-DHEA show the reversible production of 7beta-hydroxy-DHEA through an intermediary 7-oxo-DHEA. Both the production of 7alpha-hydroxysteroids and their interference with the activation of cortisone into cortisol are basic to the concept of native anti-glucocorticoids. The cytochrome P4507B1 responsible for 7alpha-hydroxy-DHEA production and 11beta-HSD1 are key enzymes for the modulation of glucocorticoid action in humans. This is a promising new area for research.

Neurosteroids: metabolism in human intestine microsomes.

Both dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA) are substrate for cytochrome P450 species and enzymes that produce 7alpha- and 7beta-hydroxylated metabolites in the brain and other organs. In contrast to DHEA and EpiA, the 7-hydroxylated derivatives were shown to mediate neuroprotection, and 7beta-hydroxy-EpiA was the most potent. The suggested use of any of these steroids as drugs administered per os for neuroprotection requires the assessment of their metabolism in the human intestine and liver. To achieve this, we produced radio-labeled 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA that were used as substrates in incubations with human intestine microsomes supplemented with reduced or oxidized cofactors. Identity of the radio-labeled metabolites obtained was determined by gas chromatography/mass spectrometry after comparison with authentic steroid references. The proportions of metabolites produced resulted from their radioactivity contents. The only metabolite obtained with DHEA, EpiA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA substrates was its 17beta-reduced derivative, thus inferring the presence of 17beta-hydroxysteroid oxidoreductases in the human intestine microsomes. In addition to the 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA substrates, their 17beta-reduced metabolites were obtained with 7beta-hydroxy-EpiA and 7alpha-hydroxy-EpiA, respectively. The identity of the enzyme responsible for the 7alpha-hydroxy-EpiA/7beta-hydroxy-EpiA inter-conversion is unknown. The incubation conditions used produced these metabolites in low but significant yields that suggest their presence in the portal blood before access to the liver.

Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.

The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues.

Human liver S9 fractions: metabolism of dehydroepiandrosterone, epiandrosterone, and related 7-hydroxylated derivatives.

Dehydroepiandrosterone (DHEA) and 3beta-hydroxy-5alpha-androstan-17-one (epiandrosterone, EpiA) are both precursors for 7alpha- and 7beta-hydroxylated metabolites in the human brain. These 7-hydroxylated derivatives were shown to exert anti-glucocorticoid and neuroprotective effects. When these steroids are administered per os to humans, the first organ encountered is the liver, where extensive metabolism takes place. The objective of this work was to assess the cofactor dependence and metabolism of DHEA, EpiA, and their 7-hydroxylated derivatives in S9 fractions of human liver, using a radiolabeled steroid substrate for quantification and gas chromatography-mass spectrometry for identification. The best transformation yields were obtained with NADPH and were larger in female than in male. Results showed that both DHEA and EpiA mainly transformed into their 17beta-hydroxylated derivatives, 7- or 16alpha-hydroxylated metabolites under NAD(P)H conditions, and 5alpha-androstane-3,17-dione for EpiA under NAD(P)+ conditions. In turn, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA were partly transformed into each other via a 7-oxo-DHEA intermediate and were reduced into the 17beta-hydroxy derivative, respectively. The same type of transformations occurred for 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA, except that no 7-oxo-EpiA intermediate was obtained. These findings determine the presence of enzymes responsible for the 7alpha- and 16alpha-hydroxylation in the human liver, the 11beta-hydroxysteroid dehydrogenase type 1 responsible for the oxidoreduction of the 7-hydroxylated substrates, and the 17beta-hydroxysteroid dehydrogenase responsible for the reduction of 17-oxo-steroids into 17beta-hydroxysteroids.

The human cytochrome P4507B1: catalytic activity studies.

The cytochrome P4507B1 (P4507B1) in the human hippocampus is responsible for the production of 7alpha-hydroxylated derivatives of dehydroepiandrosterone (DHEA) and other 3beta-hydroxylated neurosteroids. Minor quantities of the 7beta-hydroxylated derivatives are also produced. Neuroprotective action of these 7-hydroxysteroids was reported. Recombinant human P4507B1 was prepared from yeast coexpressing the human hippocampal P450 cDNA and the human P450 reductase genes. Microsomal P4507B1 activity was tested in the presence of NADPH and (14)C-labeled steroid substrates to deduce kinetic parameters and to study inhibitor responses. The K(M) values obtained for DHEA, pregnenolone, epiandrosterone, 5alpha-androstane-3beta,17beta-diol and estrone were 1.90 +/- 0.06, 1.45 +/- 0.03, 1.05 +/- 0.12, 0.8 +/- 0.04 and 1.20 +/- 0.26 microM, respectively. Production of limited amounts of 7beta-hydroxylated derivatives was also observed, but only with DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone. K(M) values determined for 7beta-hydroxylation were identical to those for 7alpha-hydroxylation. The DHEA 7alpha-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [25-35] beta-amyloid peptide (non-competitive inhibition). These results indicate that in human, the 7-hydroxylation catalysed by P4507B1 preferentially takes place on DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone with major and minor formation of 7alpha- and 7beta-hydroxylated derivatives, respectively. Both estrogens and a beta-amyloid component inhibit the P4507B1-mediated production of the 7-hydroxysteroid metabolites.

Use of bioconversion for the preparation of [4-14C]-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone.

The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1. Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of [4-14C]-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.

Argyrophilic grain disease and Alzheimer's disease are distinguished by their different distribution of tau protein isoforms.

Prominent neuronal and glial tau filamentous inclusions are hallmarks of neurodegenerative tauopathies, among them Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and argyrophilic grain disease (AgD). AgD is a late onset dementia in which pathologically aggregated tau proteins are found in limbic structures in the shape of distinct argyrophilic grains and coiled bodies. Until now tau protein deposits in AgD have not been assessed biochemically. We therefore decided to investigate the electrophoretic profile of pathological tau protein as well as the tau protein isoform composition of filamentous inclusions in AgD cases. A distinct pathological tau doublet at 64 and 69 kDa and a minor 74-kDa band was obtained in two AgD cases with only very mild concomitant AD pathology (Braak stage I), while in two AgD cases with moderate AD pathology (Braak stage II and III, respectively), an additional minor band at 60 kDa was detected. Thus, the pathological tau profile (PTP) in pure AgD cases differs from both the PTPs in AD (tau triplet at 60, 64 and 69 kDa, minor band at 74 kDa) and PiD (major tau doublet at 60 and 64 kDa, minor band at 69 kDa) but not from those in PSP and CBD. Using a two-dimensional gel electrophoresis approach anti-exon 10 antiserum strongly stained the AgD doublet and the minor 74-kDa band, while anti-exon 2 and 3 antisera only faintly stained the 69- and the minor 74-kDa component, thus suggesting that pathological tau aggregates in AgD are mainly made of four-repeat (4R) tau isoforms. Furthermore, in contrast to earlier immunohistochemical studies, we now show biochemically that Ser262 indeed is phosphorylated in the PTP of AgD. Finally, expression of normal tau protein was not found to be altered in AgD. Altogether, our results demonstrate that AgD is characterized by a major tau doublet that is distinct from AD and PiD. AgD, however, shares the pathological tau doublet (64 and 69 kDa) as well as the predominance of 4R tau isoforms with CBD and PSP.