Analytical validation of ONCURIA™ a multiplex bead-based immunoassay for the non-invasive bladder cancer detection.

BACKGROUND: The objective of our study was to assess the analytical performance of a multiplex assay (Oncuria™) to quantify protein biomarkers towards a bladder cancer associated diagnostic signature in voided urine. METHOD: ology: Using Luminex xMAP technology, a custom immunoassay was developed to measure the concentrations of 10 urinary analytes (angiogenin, ANG; apolipoprotein E, APOE; alpha-1 antitrypsin, A1AT; carbonic anhydrase 9, CA9; interleukin 8, IL8; matrix metallopeptidase 9, MMP9; matrix metallopeptidase 10, MMP10; plasminogen activator inhibitor 1, PAI1; syndecan 1, SDC1; vascular endothelial growth factor, VEGF). Selectivity, sensitivity, specificity, precision, linearity, dynamic range, and detection threshold were assessed using recombinant proteins and human urine samples. Analytical variability with respect to batch size, run, day, operator, and interference were also evaluated. RESULTS: Analytical evaluation demonstrated a) all antigen cross-reactivity was noted to be <1% of the tested concentration, b) minimal detected dose ranged from 0.295 â€‹pg/mL in IL8 to 31.1 â€‹pg/mL in APOE, c) highly reproducible and accurate noting coefficient of variation (CV) and relative error (RE) values below 15% for all analytes and d) minimal interference. The assay can be completed in <5 â€‹h using as little as 150 â€‹µL of voided urine. CONCLUSION: To our knowledge, this is the first multiplex bead-based immunoassay for the non-invasive detection of bladder cancer that has been analytically validated as a tool with the potential to help clinicians manage patients at risk of harboring bladder cancer.

Caspase-2 cleavage of tau reversibly impairs memory.

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

Soluble Conformers of Aß and Tau Alter Selective Proteins Governing Axonal Transport.

UNLABELLED: Despite the demonstration that amyloid-ß (Aß) can trigger increased tau phosphorylation and neurofibrillary tangle (NFT) formation in vivo, the molecular link associating Aß and tau pathologies remains ill defined. Here, we observed that exposure of cultured primary neurons to Aß trimers isolated from brain tissue of subjects with Alzheimer's disease led to a specific conformational change of tau detected by the antibody Alz50. A similar association was supported by postmortem human brain analyses. To study the role of Aß trimers in vivo, we created a novel bigenic Tg-Aß+Tau mouse line by crossing Tg2576 (Tg-Aß) and rTg4510 (Tg-Tau) mice. Before neurodegeneration and amyloidosis, apparent Aß trimers were increased by ∼2-fold in 3-month-old Tg-Aß and Tg-Aß+Tau mice compared with younger mice, whereas soluble monomeric Aß levels were unchanged. Under these conditions, the expression of soluble Alz50-tau conformers rose by ∼2.2-fold in the forebrains of Tg-Aß+Tau mice compared with nontransgenic littermates. In parallel, APP accumulated intracellularly, suggestive of a putative dysfunction of anterograde axonal transport. We found that the protein abundance of the kinesin-1 light chain (KLC1) was reduced selectively in vivo and in vitro when soluble Aß trimers/Alz50-tau were present. Importantly, the reduction in KLC1 was prevented by the intraneuronal delivery of Alz50 antibodies. Collectively, our findings reveal that specific soluble conformers of Aß and tau cooperatively disrupt axonal transport independently from plaques and tangles. Finally, these results suggest that not all endogenous Aß oligomers trigger the same deleterious changes and that the role of each assembly should be considered separately. SIGNIFICANCE STATEMENT: The mechanistic link between amyloid-ß (Aß) and tau, the two major proteins composing the neuropathological lesions detected in brain tissue of Alzheimer's disease subjects, remains unclear. Here, we report that the trimeric Aß species induce a pathological modification of tau in cultured neurons and in bigenic mice expressing Aß and human tau. This linkage was also observed in postmortem brain tissue from subjects with mild cognitive impairment, when Aß trimers are abundant. Further, this modification of tau was associated with the intracellular accumulation of the precursor protein of Aß, APP, as a result of the selective decrease in kinesin light chain 1 expression. Our findings suggest that Aß trimers might cause axonal transport deficits in AD.

Amyloid plaque and neurofibrillary tangle pathology in a regulatable mouse model of Alzheimer's disease.

Transgenic mouse models that independently express mutations in amyloid precursor protein (APP) and tau have proven useful for the study of the neurological consequences of amyloid-beta (Abeta) plaque and neurofibrillary tangle pathologies. Studies using these mice have yielded essential discoveries with regard to specific aspects of neuronal dysfunction and degeneration that characterize the brain during Alzheimer's disease (AD) and other age-dependent tauopathies. Most recent transgenic studies have focused on the creation of regulatable models that allow the temporal control of transgene expression. To study a more complete model of AD pathology, we designed a new regulatable transgenic mouse that harbors both APP and tau transgenes. Here, we present a novel transgenic mouse model, rTg3696AB, which expresses human APP(NLI) and tau(P301L) driven by the CaMKII promoter system. Subsequent generation of Abeta and 4R0N tau in the brain resulted in the development of three neuropathological features of AD: Abeta plaques, neurofibrillary tangles, and neurodegeneration. Importantly, transgene expression in these mice is regulatable, permitting temporal control of gene expression and the investigation of transgene suppression.

Androgens regulate neprilysin expression: role in reducing beta-amyloid levels.

Age-related testosterone depletion in men is a risk factor for Alzheimer's disease. Prior studies suggest that androgens affect Alzheimer's disease risk by regulating accumulation of beta-amyloid protein (Abeta) by an undefined mechanism. In this study, we investigated the role of the Abeta-catabolizing enzyme neprilysin (NEP) in this process. First, we observed that androgens positively regulate neural expression of NEP in adult male rats. Next, we investigated androgen regulatory effects on both NEP expression and Abeta levels using cultured hippocampal neurons and neuronally differentiated rat pheochromocytoma cell 12 with or without androgen receptor (AR). Dihydrotestosterone (DHT) induced a time-dependent increase in NEP expression. DHT also significantly decreased levels of Abeta in AR-expressing cells transfected with amyloid precursor protein, but did not affect levels of either full-length or non-amyloidogenic, soluble amyloid precursor protein. Importantly, the DHT induced decrease of Abeta was blocked by pharmacological inhibition of NEP. The DHT-mediated increase in NEP expression and decrease in Abeta levels were (i) not observed in rat pheochromocytoma cell 12 lacking AR and (ii) blocked in AR-expressing cells by the antagonists, cyproterone acetate and flutamide. Together, these findings suggest that androgen regulation of Abeta involves an AR-dependent mechanism requiring up-regulation of the Abeta catabolizing enzyme NEP.

Androgen cell signaling pathways involved in neuroprotective actions.

As a normal consequence of aging in men, testosterone levels significantly decline in both serum and brain. Age-related testosterone depletion results in increased risk of dysfunction and disease in androgen-responsive tissues, including brain. Recent evidence indicates that one deleterious effect of age-related testosterone loss in men is increased risk for Alzheimer's disease (AD). We discuss recent findings from our laboratory and others that identify androgen actions implicated in protecting the brain against neurodegenerative diseases and begin to define androgen cell signaling pathways that underlie these protective effects. Specifically, we focus on the roles of androgens as (1) endogenous negative regulators of beta-amyloid accumulation, a key event in AD pathogenesis, and (2) neuroprotective factors that utilize rapid non-genomic signaling to inhibit neuronal apoptosis. Continued elucidation of cell signaling pathways that contribute to protective actions of androgens should facilitate the development of targeted therapeutic strategies to combat AD and other age-related neurodegenerative diseases.

Progestins inhibit the neuroprotective effects of estrogen in rat hippocampus.

Although estrogen has beneficial actions in brain, recent clinical trials demonstrated adverse neural effects of hormone therapy in postmenopausal women. The cause(s) of this disconnect between experimental and clinical findings may include unanticipated effects of progestins. We report that both natural progesterone and the clinical progestin medroxyprogesterone acetate block estrogen neuroprotection. These findings underscore the need to evaluate neural actions of progestins in the rational design of hormone therapy.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

A central role for ROS in the functional remodelling of L-type Ca2+ channels by hypoxia.

Periods of prolonged hypoxia are associated clinically with an increased incidence of dementia, the most common form of which is Alzheimer's disease. Here, we review recent studies aimed at providing a cellular basis for this association. Hypoxia promoted an enhanced secretory response of excitable cells via formation of a novel Ca2+ influx pathway associated with the formation of amyloid peptides of Alzheimer's disease. More strikingly, hypoxia potentiated Ca2+ influx specifically through L-type Ca2+ channels in three distinct cellular systems. This effect was post-transcriptional, and evidence suggests it occurred via increased formation of amyloid peptides which alter Ca2+ channel trafficking via a mechanism involving increased production of reactive oxygen species by mitochondria. This action of hypoxia is likely to contribute to dysregulation of Ca2+ homeostasis, which has been proposed as a mechanism of cell death in Alzheimer's disease. We suggest, therefore, that our data provide a cellular basis to account for the known increased incidence of Alzheimer's disease in patients who have suffered prolonged hypoxic episodes.

Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L).

Here, we describe the generation of a novel transgenic mouse model of human tauopathy. The rTg(tau(P301L))4510 mouse expresses the P301L mutation in tau (4R0N) associated with frontotemporal dementia and parkinsonism linked to chromosome 17. Transgene expression was driven by a forebrain-specific Ca(2+) calmodulin kinase II promoter system resulting in high levels of expression in the hippocampus and neocortex. Importantly, transgene expression in this model is induced via the tetracycline-operon responsive element and is suppressed after treatment with doxycycline. Continued transgene expression in rTg(tau(P301L))4510 mice results in age-dependent development of many salient characteristics of hereditary human dementia. From an early age, immunohistochemical studies demonstrated abnormal biochemical processing of tau and the presence of pathological conformation- and phosphorylation-dependent epitopes. Neurofibrillary tangle (NFT) pathology was first observed in the neocortex and progressed into the hippocampus and limbic structures with increasing age. Consistent with the formation of NFTs, immunoblots indicated an age-dependent transition of accumulating tau species from Sarkosyl soluble 55 kDa to insoluble hyperphosphorylated 64 kDa. Ultrastructural analysis revealed the presence of straight tau filaments. Furthermore, the effects of tau(P301L) expression on spatial reference memory were longitudinally tested using the Morris water maze. Compared with nontransgenic age-matched control littermates, rTg(tau(P301L))4510 mice developed significant cognitive impairments from 4 months of age. Memory deficits were accompanied by gross forebrain atrophy and a prominent loss of neurons, most strikingly in hippocampal subdivision CA1. Collectively, these data describe a novel transgenic mouse that closely mimics human tauopathy and may represent an important model for the future study of tau-related neurodegenerative disease.

Exercise increases the vulnerability of rat hippocampal neurons to kainate lesion.

Available evidence suggests that regular, moderate-intensity exercise has beneficial effects on neural health, perhaps including neuroprotection. To evaluate this idea further, we compared the severity of kainate-induced neuronal loss in exercised versus sedentary female rats. Stereological estimations of neuron number revealed that rats in the exercise condition exhibited significantly greater neuron loss in hippocampal region CA2/3, suggesting that high levels of physical activity may increase neuronal vulnerability to excitotoxicity.

Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid beta protein (1-40) is dependent on solubility status.

The Alzheimer's disease peptide amyloid beta protein (Abeta) can exist in soluble and fibrillar, aggregated forms. Abeta in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca(2+) homeostasis. This process may involve changes in Ca(2+) influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Abeta to cultured rat cortical neurones. Unaggregated Abeta(1-40) (24-h pretreatment, 1 microM) stimulated an increase in voltage-dependent Ca(2+) channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Abeta(1-40) pre-treatment reduced Ca(2+) channel current density in cortical neurones via an action on N-type Ca(2+) current. This failure of aggregated Abeta(1-40) to increase the Ca(2+) channel current was confirmed on cerebellar granule neurone Ca(2+) currents which normally undergo an increase in activity following soluble Abeta application. Using the MTT and TUNEL assays, aggregated Abeta(1-40) was found to promote apoptotic cell death in cortical neurones confirming that Abeta exhibited the expected biological activity. Unaggregated Abeta had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Abeta(1-40), and not the aggregated form, cause changes in neuronal Ca(2+) channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.