First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media.

The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant differences for sperm viability and total motility were observed between the two extenders, as was the concentration of ProAKAP4 both at T0 and T4. At T4, all quality parameters and ProAKAP4 content significantly decreased compared to T0, but with a considerably slower decrease in one extender than the other. These preliminary results suggest that measuring the concentration of ProAKAP4 is a promising tool for the comparison of different extenders and the selection of the optimal freezing medium for each stallion ejaculate.

Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa.

A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentration of proAKAP4 measured by ELISA in post-thawed semen. Frozen sperm from 13 different stallions were used. Semen samples (n = 17) were prepared using the INRA Freeze medium to reach a concentration of 150 million spermatozoa/mL, packaged and frozen in 0.5 mL straws. The precursor proAKAP4 and the mature protein AKAP4 both localize to the fibrous sheath of the principle piece of equine sperm flagellum. The concentrations of proAKAP4 were determined in the post-thawed semen using ELISA method (Horse 4MID® kits, 4BioDx, France). The mean concentration of proAKAP4 was then of 7.372 ±â€¯0.79 ng/µL and was significantly correlated with the post-thawed total motility (Pearson coefficient r = 0.66, p = 0.002) and progressive motility (Pearson coefficient r = 0.76, p = 0.0002) and the amount of proAKAP4 represent the amount of spermatozoa that expressed proAKAP4. Taken together, these preliminary results confirm the interest to use proAKAP4 concentrations as a promising marker of stallion sperm quality as close correlation was observed between the proAKAP4 concentration and sperm motility parameters.

A-kinase anchor protein 4 precursor (pro-AKAP4) in human spermatozoa.

BACKGROUND: A-kinase anchor protein 4 (AKAP4) and its precursor pro-AKAP4 are two major proteins in spermatozoa of rodents and mammals. Although researchers have characterized the AKAP4 expression in various species, the protein's expression in humans has not been described in detail. OBJECTIVES: The objective of this study was to characterize human pro-AKAP4 more precisely (notably the definition of its localization and expression levels in human spermatozoa and testes). MATERIALS AND METHODS: pro-AKAP4 protein expression levels were assessed by Western blotting. The pro-AKAP4's localization in spermatozoa and testes was determined using immunofluorescence staining and immunogold electron microscopy. Furthermore, pro-AKAP4 protein expression levels were assessed in a series of 77 human semen samples, and associations with semen parameters were evaluated. RESULTS: Western blotting revealed a 100-kDa band in human sperm protein extracts. The pro-AKAP4 was immunolocalized in the fibrous sheath of the flagellum of ejaculated spermatozoa and in elongated spermatids in human testes. A Western blot analysis of 77 normozoospermic semen samples evidenced striking differences in pro-AKAP4 levels from one to another sample (median [interquartile range] integrated optical density = 305 [49-1038]). No correlations were found for pro-AKAP4 levels on one hand and semen volume, sperm concentration, sperm count, sperm motility, or sperm morphology on the other (p > 0.05 for all). However, pro-AKAP4 levels were positively correlated with motility after density gradient centrifugation of the semen (r = 0.224, p = 0.049). DISCUSSION: AKAP4 protein might be activated as an alternative pathway to rescue sperm motility. In human spermatozoa, pro-AKAP4 might therefore be a 'reservoir' of mature AKAP4. CONCLUSION: This study generated new knowledge about pro-AKAP4 in human semen, which may be of interest in the management of male infertility.

Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology.

A recent genome-wide association meta-analysis for Alzheimer's disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of >6000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice. These data indicate that PTK2B acts as an early marker and in vivo modulator of Tau toxicity.

A2A adenosine receptor deletion is protective in a mouse model of Tauopathy.

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.

[The testicular microtubule-associated protein Tau: Where, when during spermatogenesis?]. / La protéine microtubulaire Tau testiculaire: une place dans la spermatogenèse?

The Tau protein (Tubulin Associated Unit) is a phosphoprotein of the microtubule-associated protein family (MAPs). Its role is the regulation of the microtubule polymerization. The Tau protein is naturally present in brain, heart, muscle, lung, kidney, pancreas and liver. An expression of Tau protein and RNA messengers was also highlighted in the testis that is an organ rich in microtubules. The role of microtubules is essential in the stabilization of the cellular shape and in cell divisions. In the testis, Tau protein could be involved in the division process of the spermatogenesis by acting on the microtubular dynamics in the arrangement of the spermatozoon polarity. This review synthesizes the current knowledge, the localization and the main functions of the Tau protein focused on the testis. The localization and the potential roles of the Tau protein during the spermatogenesis are discussed by emphasizing the link with the microtubular structures of seminiferous tubules.

Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2.

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n>50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscle blind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.

Mis-splicing of Tau exon 10 in myotonic dystrophy type 1 is reproduced by overexpression of CELF2 but not by MBNL1 silencing.

Tau is the proteinaceous component of intraneuronal aggregates common to neurodegenerative diseases called Tauopathies, including myotonic dystrophy type 1. In myotonic dystrophy type 1, the presence of microtubule-associated protein Tau aggregates is associated with a mis-splicing of Tau. A toxic gain-of-function at the ribonucleic acid level is a major etiological factor responsible for the mis-splicing of several transcripts in myotonic dystrophy type 1. These are probably the consequence of a loss of muscleblind-like 1 (MBNL1) function or gain of CUGBP1 and ETR3-like factor 1 (CELF1) splicing function. Whether these two dysfunctions occur together or separately and whether all mis-splicing events in myotonic dystrophy type 1 brain result from one or both of these dysfunctions remains unknown. Here, we analyzed the splicing of Tau exons 2 and 10 in the brain of myotonic dystrophy type 1 patients. Two myotonic dystrophy type 1 patients showed a mis-splicing of exon 10 whereas exon 2-inclusion was reduced in all myotonic dystrophy type 1 patients. In order to determine the potential factors responsible for exon 10 mis-splicing, we studied the effect of the splicing factors muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1), CUGBP1 and ETR3-like factor 2 (CELF2), and CUGBP1 and ETR3-like factor 4 (CELF4) or a dominant-negative CUGBP1 and ETR-3 like factor (CELF) factor on Tau exon 10 splicing by ectopic expression or siRNA. Interestingly, the inclusion of Tau exon 10 is reduced by CUGBP1 and ETR3-like factor 2 (CELF2) whereas it is insensitive to the loss-of-function of muscleblind-like 1 (MBNL1), CUGBP1 and ETR3-like factor 1 (CELF1) gain-of-function, or a dominant-negative of CUGBP1 and ETR-3 like factor (CELF) factor. Moreover, we observed an increased expression of CUGBP1 and ETR3-like factor 2 (CELF2) only in the brain of myotonic dystrophy type 1 patients with a mis-splicing of exon 10. Taken together, our results indicate the occurrence of a mis-splicing event in myotonic dystrophy type 1 that is induced neither by a loss of muscleblind-like 1 (MBNL1) function nor by a gain of CUGBP1 and ETR3-like factor 1 (CELF1) function but is rather associated to CUGBP1 and ETR3-like factor 2 (CELF2) gain-of-function.

Overexpression of MBNL1 fetal isoforms and modified splicing of Tau in the DM1 brain: two individual consequences of CUG trinucleotide repeats.

Neurofibrillary degeneration is often observed in the brain of patients with type 1 myotonic dystrophy (DM1). It consists principally of the aggregation of Tau isoforms that lack exon 2/3 encoded sequences, and is the consequence of the modified splicing of Tau pre-mRNA. In experimental models of DM1, the splicing of several transcripts is modified due to the loss of Muscleblind-like 1 (MBNL1) function. In the present study, we demonstrate that the MBNL1 protein is also present in the human brain, and consists of several isoforms, as shown by RT-PCR and sequencing. In comparison with controls, we show that the adult DM1 brain exhibits modifications in the splicing of MBNL1, with the preferential expression of long MBNL1 isoforms--a splicing pattern similar to that seen in the fetal human brain. In cultured HeLa cells, the presence of long CUG repeats, such as those found in the DM1 mutation, leads to similar changes in the splicing pattern of MBNL1, and the localization of MBNL1 in nuclear RNA foci. Long CUG repeats also reproduce the repression of Tau exon 2/3 inclusion, as in the human disease, suggesting that their effect on MBNL1 expression may lead to changes in Tau splicing. However, while an overall reduction in the expression of MBNL1 mimics the effect of the DM1 mutation, none of the MBNL1 isoforms tested so far modulates the endogenous splicing of Tau. The modified splicing of Tau thus results from a possibly CUG-mediated loss of function of MBNL1, but not from changes in the MBNL1 expression pattern.

Similar brain tau pathology in DM2/PROMM and DM1/Steinert disease.

Neurofibrillary degeneration (NFD) occurs in the brains of patients with myotonic dystrophy (DM) type 1. The authors report a similar tau pathology in the CNS of a patient with DM2 and compare it to that of patients with DM1. A reduced expression of tau exon 2 and exon 3 epitopes is observed in both DM1 and DM2. This suggests a similar physiopathologic process that may contribute to common neurologic features in patients with DM.

Frontotemporal dementia, motor neuron disease and tauopathy: clinical and neuropathological study in a family.

We report a familial disorder occurring in three patients that presented as frontotemporal dementia (FTD). A neuropathological study was performed in a 58-year-old patient, who developed FTD 2 years prior to the onset of motor neuron disease (MND), and died at age 62. Lesions indicative of associated MND were observed: neuronal loss in the anterior horns of the spinal cord, Bunina bodies, axonal spheroids, degeneration of the pyramidal tracts, and of FTD: decreased neuronal density and laminar microvacuolation of layers II and III in the frontal and temporal cortex. Ubiquitin-only-immunoreactive changes were found in the spinal cord and medulla, but were absent from the temporal and frontal cortex. There were also widespread deposits of various neuronal and glial inclusions containing abnormally phosphorylated tau protein, the Western blotting pattern of which was characterized by two major bands of 64 and 69 kDa. There were no abnormalities of the entire coding sequences of microtubule-associated protein tau (MAPT) and copper-zinc superoxide dismutase (SOD(1)) genes. Our results suggest that FTD associated with MND can be caused by a larger spectrum of neuropathological lesions than commonly accepted.

Tau aggregates are abnormally phosphorylated in inclusion body myositis and have an immunoelectrophoretic profile distinct from other tauopathies.

Sporadic inclusion body myositis (s-IBM) is the most frequent progressive acquired inflammatory myopathy in people older than 50 years. Abnormal aggregates of 'Alzheimer's proteins', including tau proteins, have been previously demonstrated in s-IBM. In the present study, we have investigated by immunohistochemistry and immunoblotting analysis the presence of tau proteins in muscle biopsy samples from patients with s-IBM and other myopathies with rimmed vacuoles, using newly developed antibodies raised against tau protein epitopes found in Alzheimer's disease brain. Tau immunoreactivity was shown in rimmed vacuoles or inclusions, preferentially with antibodies directed against phosphorylated carboxy-terminal epitopes of tau proteins. Cytoplasmic reactivity was also demonstrated in atrophic, nonvacuolated fibres, as well as in non-necrotic fibres invaded by inflammatory cells. Abnormally phosphorylated tau aggregates were also found in other compartments of the muscle fibre in s-IBM and other myopathies. Tau immunoblotting showed an electrophorectic profile of a doublet within the range of 60-62 kDa isovariants, which was different from tauopathies of the central nervous system. Finally, the unique pattern of immunoreactivity of s-IBM samples towards anti-tau antibodies is a new clue to a possible distinct subclass of peripheral tauopathy, different from the tauopathies of the central nervous system.

Association of ATP synthase alpha-chain with neurofibrillary degeneration in Alzheimer's disease.

Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.

Nonoverlapping but synergetic tau and APP pathologies in sporadic Alzheimer's disease.

OBJECTIVE: To determine the spatiotemporal mapping of tau pathologies and insoluble pools of Abeta in aging and sporadic AD, and their contribution to the physiopathologic, clinical, and neuropathologic features. METHODS: The authors studied 130 patients of various ages and different cognitive status, from nondemented controls (n = 60) to patients with severe definite AD (n = 70) who were followed prospectively. Insoluble Abeta 42 and 40 species were fully solubilized and quantified in the main neocortical areas, with a new procedure adapted to human brain tissue. Tau pathology staging was determined in 10 different brain areas, using Western blots. RESULTS: In AD, there is a constellation of amyloid phenotypes, extending from cases with exclusively aggregated Abeta 42 to cases with, in addition, large quantities of insoluble Abeta 40 species. Five other points were observed: 1) There was no spatial and temporal overlap in the distribution of these two insoluble Abeta species in cortical brain areas. 2) In contrast to solubilized Abeta 40 aggregates composed essentially of monomers and dimers, solubilized Abeta 42 was essentially observed as dimers and multimers. 3) Abeta 42 aggregates were observed at the early stages of tau pathology, whereas the insoluble Abeta 40 pool was found at the last stages. 4) During the progression of the disease, Abeta aggregates increase in quantity and heterogeneity, in close parallel to the extension of tau pathology. 5) There was no spatial overlap between Abeta aggregation that is widespread and heterogeneously distributed in cortical areas and tau pathology that is progressing sequentially, stereotypically, and hierarchically. CONCLUSIONS: These observations demonstrate that Abeta 42 aggregation, and not Abeta 40, is the marker that is close to Alzheimer etiology. It should be the main target for the early biological diagnosis of AD and modeling. Furthermore, the spatial mismatch between amyloid ss-precursor protein (APP) and tau pathologies in cortical brain areas demonstrates that neurodegeneration is not a direct consequence of extracellular Abeta neurotoxicity. Hence, there is a synergetic effect of APP dysfunction, revealed by Abeta aggregation, on the neuron-to-neuron propagation of tau pathology.

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.

Paradoxical phosphorylation of the serine 199 on tau proteins from young individuals.

The microtubule-associated tau proteins are abnormally aggregated in many tauopathies. Phosphorylation modulates the functions of tau. The serine 199 residue of tau is abnormally phosphorylated at early and late stages of Alzheimer's disease. The presence of the phosphorylated Ser199 was investigated in autopsy-derived and biopsy-derived brain tissue samples from non-demented individuals. A paradoxical expression was found in the hippocampus of the youngest ones, in granule cells of the dentate gyrus and in pyramidal cells of the Ammon's horn, which are particularly prone to neurodegeneration in several tauopathies. The rate of positive cells decreased with age. These data emphasize the importance of the phosphorylation of the Ser199 residue of tau in ageing and susceptibility to neurodegeneration.

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1.

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.

Proteoglycans: pericellular and cell surface multireceptors that integrate external stimuli in the mammary gland.

Proteoglycans consist of a core protein and an associated glycosaminoglycan (GAG) chain of heparan sulfate, chondroitin sulfate, dermatan sulfate or keratan sulfate, which are attached to a serine residue. The core proteins of cell surface proteoglycans may be transmembrane, e.g., syndecan, or GPI-anchored, e.g., glypican. Many different cell surface and matrix proteoglycan core proteins are expressed in the mammary gland and in mammary cells in culture. The level of expression of these core proteins, the structure of their GAG chains, and their degradation are regulated by many of the effectors that control the development and function of the mammary gland. Regulatory proteins of the mammary gland that bind GAG include many growth factors and morphogens (fibroblast growth factors, hepatocyte growth factor/scatter factor, members of the midkine family, wnts), matrix proteins (collagen, fibronectin, and laminin), enzymes (lipoprotein lipase) and microbial surface proteins. Structural diversity within GAG chains ensures that each protein-GAG interaction is as specific as necessary and a number of sequences of saccharides that recognize individual proteins have been elucidated. The GAG-protein interactions serve to regulate the signal output of growth factor receptor tyrosine kinase and hence cell fate as well as the storage and diffusion of extracellular protein effectors. In addition, GAGs clearly coordinate stromal and epithelial development, and they are active participants in mediating cell-cell and cell-matrix interactions. Since a single proteoglycan, even if it carries a single GAG chain, can bind multiple proteins, proteoglycans are also likely to act as multireceptors which promote the integration of cellular signals.