ABSTRACT
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.
Subject(s)
Alzheimer Disease , Tauopathies , Animals , Humans , Mice , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Biomarkers/metabolism , Chromatography, Liquid , Phosphorylation , Serine/metabolism , Tandem Mass Spectrometry , tau Proteins/metabolism , Tauopathies/metabolism , Trypsin/metabolismABSTRACT
Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Fibroblast Growth Factor 2/pharmacology , Neural Cell Adhesion Molecules/pharmacology , Neurites/physiology , Neurons/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Adaptor Proteins, Signal Transducing/drug effects , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Cells, Cultured , Coculture Techniques , GRB2 Adaptor Protein , Humans , Membrane Proteins/physiology , Mice , Neurites/drug effects , Neurons/drug effects , Neurons/ultrastructure , PC12 Cells , Phosphoproteins/physiology , Phosphorylation/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , Rats , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , src-Family Kinases/metabolismABSTRACT
The neural cell adhesion molecule (NCAM) plays a pivotal role in neural development, regeneration, and plasticity. NCAM mediates adhesion and subsequent signal transduction through NCAM-NCAM binding. Recently, a peptide ligand termed P2 corresponding to a 12-amino-acid sequence in the FG loop of the second Ig domain of NCAM was shown to mimic NCAM homophilic binding as reflected by induction of neurite outgrowth in hippocampal neurons. We demonstrate here that in concentrations between 0.1 and 10 microM, P2 also induced neuritogenesis in primary dopaminergic and cerebellar neurons. Furthermore, it enhanced the survival rate of cerebellar neurons although not of mesencephalic dopaminergic neurons. Moreover, our data indicate that the protective effect of P2 in cerebellar neurons was due to an inhibition of the apoptotic process, in that caspase-3 activity and the level of DNA fragmentation were lowered by P2. Finally, treatment of neurons with P2 resulted in phosphorylation of the ser/thr kinase Akt. Thus, a small peptide mimicking homophilic NCAM interaction is capable of inducing differentiation as reflected by neurite outgrowth in several neuronal cell types and inhibiting apoptosis in cerebellar granule neurons.
Subject(s)
Myelin Proteins/pharmacology , Neural Cell Adhesion Molecules/metabolism , Neurites/drug effects , Neurons/drug effects , Protein Serine-Threonine Kinases , Tyrosine 3-Monooxygenase/metabolism , Animals , Animals, Newborn , Caspase 3 , Caspases/metabolism , Cell Survival/drug effects , Cerebellum/cytology , Cerebellum/drug effects , Dose-Response Relationship, Drug , Embryo, Mammalian , Female , GAP-43 Protein/metabolism , Glial Cell Line-Derived Neurotrophic Factor , In Situ Nick-End Labeling , Insulin-Like Growth Factor I/pharmacology , Male , Mesencephalon/cytology , Mesencephalon/embryology , Mesencephalon/growth & development , Nerve Growth Factors/pharmacology , Neurons/metabolism , Oxidopamine/pharmacology , Pregnancy , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Rats, Wistar , Sympatholytics/pharmacologyABSTRACT
The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.
