A Quantitative LC-MS/MS Method for Distinguishing the Tau Protein Forms Phosphorylated and Nonphosphorylated at Serine-396.

Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer's Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches. Here, we present an indirect LC-MS/MS method for quantification of pS396-tau. We take advantage of the reproducible miscleavage caused by S396 being preceded by a lysine (K395) and the proteolytic enzyme trypsin not cleaving when the following amino acid is phosphorylated. Therefore, treatment with trypsin discriminates between the forms of tau with and without phosphorylation at S396 and pS396-tau can be quantified as the difference between total S396-tau and nonphosphorylated S396-tau. To qualify the method, it was successfully applied for quantification of pS396-tau in human CSF from healthy controls and patients with Mild Cognitive Impairment and AD. In addition, the method was applied for rTg4510 mice where a clear dose dependent decrease in pS396-tau was observed in CSF following intravenous administration of a monoclonal antibody (Lu AF87908, hC10.2) targeting the tau epitope containing pS396. Finally, a formal validation of the method was conducted. In conclusion, this sensitive LC-MS/MS-based method for measurement of pS396-tau in CSF allows for quantitative translational biomarker applications for tauopathies including investigations of potential drug induced effects.

Highly Specific and Sensitive Target Binding by the Humanized pS396-Tau Antibody hC10.2 Across a Wide Spectrum of Alzheimer's Disease and Primary Tauopathy Postmortem Brains.

BACKGROUND: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer's disease (AD). OBJECTIVE: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. METHODS: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. RESULTS: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. CONCLUSION: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.

Antibody-mediated clearance of tau in primary mouse microglial cultures requires Fcγ-receptor binding and functional lysosomes.

Neurodegenerative diseases such as Alzheimer's disease are characterized by the progressive spreading and accumulation of hyper-phosphorylated tau protein in the brain. Anti-tau antibodies have been shown to reduce tau pathology in in vivo models and antibody-mediated clearance of tau exerted by microglia has been proposed as a contributing factor. By subjecting primary microglia cultured in vitro to anti-phospho-tau antibodies in complex with pathological tau, we show that microglia internalise and degrade tau in a manner that is dependent on FcγR interaction and functional lysosomes. It has recently been discussed if anti-tau antibody effector-functions are required for induction of tau clearance. Using antibodies with compromised FcγR binding and non-compromised control antibodies we show that antibody effector functions are required for induction of microglial clearance of tau. Understanding the inflammatory consequences of targeting microglia using therapeutic antibodies is important when developing these molecules for clinical use. Using RNA sequencing, we show that treatment with anti-tau antibodies increases transcription of mRNA encoding pro-inflammatory markers, but that the mRNA expression profile of antibody-treated cells differ from the profile of LPS activated microglia. We further demonstrate that microglia activation alone is not sufficient to induce significant tau clearance.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

Internalization of tau antibody and pathological tau protein detected with a flow cytometry multiplexing approach.

INTRODUCTION: Tau immunotherapy has emerged as a promising approach to clear tau aggregates from the brain. Our previous findings suggest that tau antibodies may act outside and within neurons to promote such clearance. METHODS: We have developed an approach using flow cytometry, a human neuroblastoma cell model overexpressing tau with the P301L mutation, and paired helical filament (PHF)-enriched pathologic tau to effectively screen uptake and retention of tau antibodies in conjunction with PHF. RESULTS: The flow cytometry approach correlates well with Western blot analysis to detect internalized antibodies in naïve and transfected SH-SY5Y cells (r2 = 0.958, and r2 = 0.968, P = .021 and P = .016, respectively). In transfected cells, more antibodies are taken up/retained as pathologic tau load increases, both under co-treated conditions and when the cells are pretreated with PHF before antibody administration (r2 = 0.999 and r2 = 0.999, P = .013 and P = .011, respectively). DISCUSSION: This approach allows rapid in vitro screening of antibody uptake and retention in conjunction with pathologic tau protein before more detailed studies in animals or other more complex model systems.

Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants.

BACKGROUND: Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary elements that can prevent enhancer-promoter interactions, if placed between these elements, and protect transgene cassettes from silencing and positional effects. It has been suggested that insulators can improve the safety and performance of lentiviral vectors. Therefore insulators have been incorporated into lentiviral vectors in order to enhance their safety profile and improve transgene expression. Commonly such insulator vectors are produced at lower titers than control vectors thus limiting their potential use. RESULTS: In this study we cloned in tandem copies of the chicken beta-globin insulator (cHS4) on both sides of the transgene cassette in order to enhance the insulating effect. Our insulator vectors were produced at significantly lower titers compared to control vectors, and we show that this reduction in titer is due to a block during the transduction process that appears after reverse transcription but before integration of the viral DNA. This non-integrated viral DNA could be detected by PCR and, importantly, prevented efficient transduction of target cells. CONCLUSION: These results have importance for the future use of insulator sequences in lentiviral vectors and might limit the use of insulators in vectors for in vivo use. Therefore, a careful analysis of the optimal design must be performed before insulators are included into clinical lentiviral vectors.

Evidence for disease-regulated transgene expression in the brain with use of lentiviral vectors.

In this study we have developed and validated a novel approach of transgene regulation in the brain. By using lentiviral vectors that incorporate promoters of genes that are up-regulated during different pathological states, we were able to regulate transgene expression in accordance with the disease process. When using a glial fibrillary acidic protein promoter, efficient disease regulation in glial cells was achieved after an excitotoxic lesion or a 6-hydroxydopamine (6-OHDA) lesion. Transgene expression was physiologically regulated and displayed a dose-dependent increase depending on the severity of lesion. Efficient regulation was also achieved in neurons when using a preproenkephlin promoter in 6-OHDA-lesioned rats, allowing combined regulation and targeting. This disease-regulated approach allows control of transgene expression in the brain without the use of inducer molecules and without overexpression of transactivator proteins.

Inhibition of chromatin condensation prevents transgene silencing in a neural progenitor cell line transplanted to the rat brain.

The use of ex vivo gene therapy in the central nervous system has so far suffered from transgene downregulation. Condensation of the transgenic sequences has been proposed to be a mechanism involved in this silencing. In this study we inhibited either histone deacetylation or DNA methylation in neural progenitor cell lines, transduced with a lentiviral vector carrying green fluorescent protein (GFP), prior to grafting them into the rat striatum. The expression of GFP was significantly higher in grafts pretreated with either of the inhibitors. After 1 week in vivo we detected an 11-fold increase in the number of GFP-expressing cells due to the inhibition of DNA methylation in vitro with azadeoxycytidine and a ninefold increase when inhibiting histone deacetylation with trichostatin A. This suggests that a pretreatment paradigm could be used to increase efficacy of ex vivo delivery of a therapeutic protein locally in the brain.

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator.

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.

Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetylation.

BACKGROUND: Ex vivo gene therapy in the central nervous system (CNS) holds great promise for diseases such as the neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. This study reports the establishment of an in vitro model of transgene down-regulation in cells grafted to the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B. METHODS: Neural cell lines were transduced at 33 degrees C with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. Cell differentiation in vitro was obtained by culturing the cells at 37 degrees C in serum-free defined media, which halts cell division, and GFP-expression was analysed by FACS. As early as day 3 of culture at 37 degrees C, the transgene expression decreased markedly in most cell lines. To validate the assay, the same clones were grafted to the adult rat striatum and the down-regulation of GFP-expression was evaluated. RESULTS: The temporal pattern of down-regulation was found to be similar in vitro and in vivo. Using this assay, it was shown that addition of inhibitors of histone deacetylation, but not an inhibitor of DNA methylation, reversed the silencing of GFP in quiescent neural progenitors by up to 308% of control values. CONCLUSION: These results suggest that the same mechanisms controlling gene transcription of the host cell's genome are active in controlling transgene expression and that this should be taken into account when constructing vectors for gene therapy. The assay reported in this study could be used as a screening method to evaluate new vectors.