Microtubule-associated Tau proteins belong to a family of factors that polymerize tubulin dimers and stabilize microtubules. Tau is strongly expressed in neurons, localized in the axon and is essential for neuronal plasticity and network. From the very beginning of Tau discovery, proteomics methods have been essential to the knowledge of Tau biochemistry and biology. In this review, we have summarized the main contributions of several proteomic methods in the understanding of Tau, including expression, post-translational modifications and structure, in both physiological and pathophysiological aspects. Finally, recent advances in proteomics technology are essential to develop further therapeutic targets and early predictive and discriminative diagnostic assays for Alzheimer's disease and related disorders.

Intraneuronal aggregates of hyperphosphorylated tau proteins, referred to as pathological tau, are found in brain areas of demented patients affected by numerous different neurodegenerative disorders. We previously described a particular biochemical profile of pathological tau proteins in myotonic dystrophy type 1 (DM1). This multisystemic disorder is characterized by an unstable CTG repeat expansion in the 3'-untranslated region of the DM protein kinase gene. In the human central nervous system, tau proteins consist of six isoforms that differ by the presence or absence of the alternatively spliced exons 2, 3 and 10. Here we show that the pattern of tau isoforms aggregated in DM1 brain lesions is characteristic. It consists mainly of the aggregation of the shortest human tau isoform. A disruption in normal tau isoform expression consisting of a reduced expression of tau isoforms containing the exon 2 was observed at both the mRNA and protein levels. Large expanded CTG repeats were detected and showed marked somatic heterogeneity between DM1 cases and in cortical brains regions analysed. Our data suggest a relationship between the CTG repeat expansion and the alteration of tau expression showing that DM1 is a peculiar tauopathy.

The role of immune responses in the cognitive impairments associated with tauopathy is unclear. Laurent et al. identify a CD8+ T-cell infiltration in the hippocampus of THY-Tau22 transgenic mice. T-cell depletion reverses spatial memory deficits in these animals, supporting a role for hippocampal T-cell infiltration in tau-driven cognitive impairments.

Plasma Abeta peptide concentrations and Abeta(1-42)/Abeta(1-40) and Abeta(n-42)/Abeta(n-40) ratios may be useful markers to indicate individuals susceptible to short-term risk of dementia.

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimer's disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.Splicing of pre-mRNA is a key post-transcriptional step in eukaryotic gene expression. A vast majority of vertebrate pre-mRNAs is alternatively spliced, allowing the production of several protein isoforms from transcripts of a given gene (1). The regulation of alternative splicing plays a major role in cell differentiation and in development and depends on the expression and activity of numerous splicing regulatory factors that are expressed differentially during development, according to the type of tissue. Defects in these alternative-splicing processes can contribute to pathogenesis, as demonstrated for a growing number of diseases, including neuromuscular diseases such as myotonic dystrophy type 1 (DM1) 9 (2, 3). DM1 is an autosomal disorder caused by an unstable CTG repeat expansion in the 3Ј-untranslated region (UTR) of the DMPK gene (4 -6). One of the main etiological hypotheses of DM1 is based on a toxic RNA gain of function, leading to the dysregulation of alternative splicing. Mutant transcripts bearing long-CUG repeats acquire unusual A-form doublestranded RNA structures (7), accumulate in the nucleus, and lead to small ribonucleoprotein inclusions, named foci (8) that sequester RNA-binding proteins such as Muscleblind-like 1 * This work was supported by the Association Française contre les Myopathies Grants 14269 and 15047, the Agence Nationale de Recherche NeuroSplicedeTau BLAN 1114 01, the EURASNET EU Contract FP6, life sciences, genomics and biotechnology for health, the Centre National de la Recherche Scientifique, the Institut National pour la Santé et la Recherche Mé dicale, the French Ministry for Youth, National Education and Research, and the Lorraine Region. The abbreviations used are: DM1, myotonic dystrophy type...

Abstract. Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimer's disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Tau's molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tauspecific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear 1 These authors jointly directed this work. N.R. Barthélemy et al. / Tau Cerebrospinal Fluid Molecular Profilepalsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

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