Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Inserm UMR 837

Looking at cholinesterases (ChEs) changes in age-related mental impairment, the expression of ChEs in brain of senescence accelerated-resistant (SAMR1) and senescence accelerated-prone (SAMP8) mice was studied. Acetylcholinesterase (AChE) activity was unmodified and BuChE activity increased twofold in SAMP8 brain. SAMR1 brain contained many AChE-T mRNAs, less BuChE and PRiMA mRNAs and scant AChE-R and AChE-H mRNAs. Their content unchanged in SAMP8 brain. Amphiphilic (G(4)(A)) and hydrophilic (G(4)(H)) AChE and BuChE tetramers, besides amphiphilic dimers (G(2)(A)) and monomers (G(1)(A)) were identified in SAMR1 brain and their distribution was little modified in SAMP8 brain. Blood plasma does not seem to provide the excess of BuChE activity in SAMP8 brain; it probably arises from glial cell changes owing to astrocytosis.

The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated mouse SAMP8.

Cylindroma of the external auditory canal: histopathology diagnostic.

The early-onset, irreversible, severe deficits of learning and memory in the senescence-accelerated mouse (SAM)-prone/8 (SAMP8) support its use as an animal model for human dementias of early onset. Possible implication of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in cognitive dysfunction of SAMP8 mice was studied by comparing cholinesterase (ChE) expression in brains of SAMP8 mice and of their normal control, SAM-resistant/1 (SAMR1) mice. The level of ChE mRNAs was the same in SAMP8 and SAMR1 brains, which agreed with their equal AChE activity (3.09 +/- 1.45 vs. 3.07 +/- 1.44 mumol.hr(-1).mg protein(-1), U/mg), but not with a doubled BuChE activity in SAMP8 brain (0.14 +/- 0.05 vs. 0.07 +/- 0.02 U/mg; P < 0.01). This great increase in neural BuChE activity may contribute to cognitive deficit of SAMP8 mice. Hydrophilic (G(4) (H), 8%) and amphiphilic (G(4) (A), 74%) AChE tetramers, besides dimers and monomers (G(2) (A) + G(1) (A), 18%), were identified in SAMR1 brains. They also contained G(4) (H) BuChE forms (18%) as well as G(4) (A) (53%) and G(2) (A) + G(1) (A) (29%) species. Although SAMP8 brain displayed proportions of AChE and BuChE forms that were similar to those of SAMR1 brain, phenyl-agarose chromatography with detergent-free extracts showed a rise in the proportion of secretory G(4) (H) BuChE from 35% in SAMR1 to 44% in SAMP8 brain. The strong immunolabelling of glial fibrillary acidic protein (GFAP), a marker of reactive gliosis, in SAMP8 brain and the consideration of BuChE as a marker of glial cells suggest a relationship between phenotypic changes in neuroglial cells and the excess of BuChE activity in SAMP8 brain.

Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8.

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.

Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of Muscleblind-like-1 isoforms.

Tau pathology is encountered in many neurodegenerative disorders known as tauopathies, including Alzheimer's disease. Physical activity is a lifestyle factor affecting processes crucial for memory and synaptic plasticity. Whether long-term voluntary exercise has an impact on Tau pathology and its pathophysiological consequences is currently unknown. To address this question, we investigated the effects of long-term voluntary exercise in the THY-Tau22 transgenic model of Alzheimer's disease-like Tau pathology, characterized by the progressive development of Tau pathology, cholinergic alterations and subsequent memory impairments. Three-month-old THY-Tau22 mice and wild-type littermates were assigned to standard housing or housing supplemented with a running wheel. After 9 months of exercise, mice were evaluated for memory performance and examined for hippocampal Tau pathology, cholinergic defects, inflammation and genes related to cholesterol metabolism. Exercise prevented memory alterations in THY-Tau22 mice. This was accompanied by a decrease in hippocampal Tau pathology and a prevention of the loss of expression of choline acetyltransferase within the medial septum. Whereas the expression of most cholesterol-related genes remained unchanged in the hippocampus of running THY-Tau22 mice, we observed a significant upregulation in mRNA levels of NPC1 and NPC2, genes involved in cholesterol trafficking from the lysosomes. Our data support the view that long-term voluntary physical exercise is an effective strategy capable of mitigating Tau pathology and its pathophysiological consequences.

Beneficial effects of exercise in a transgenic mouse model of Alzheimer's disease-like Tau pathology.

There is a growing interest in the involvement of anesthetic agents in the etiology of postoperative cognitive dysfunction. Recent animal studies suggest that acute anesthesia induces transient hyperphosphorylation of tau, an effect essentially ascribed to hypothermia. The main aim of the present study was to investigate effects, in normothermic conditions, of acute or repeated exposure to sevoflurane, a halogenated anesthetic agent, on hippocampal tau phosphorylation and spatial memory in adult mice.

Tau phosphorylation and sevoflurane anesthesia: an association to postoperative cognitive impairment.

MAPT mutations cause autosomal dominant frontotemporal lobar degeneration. These diseases are characterized by considerable heterogeneity in their clinical, neuropathological, and biochemical presentations. We describe the full characterization of a family with autosomal dominant frontotemporal lobar degeneration caused by a novel MAPT mutation. Clinical, imaging, neuropathological, and biochemical data are presented. The proband was a woman who died at 85 years old, 25 years after the onset of a slowly progressive and isolated anarthria and opercular syndrome. The pathological examination of her brain showed marked atrophy of primary motor and premotor cortices, associated with predominant neuronal tau-positive lesions mimicking Pick bodies. At the biochemical level, the six tau isoforms aggregate to display a pathological triplet at 60, 64, and 69 kDa. Two of her sons presented at 48 and 50 years old with a right temporal variant of frontotemporal degeneration characterized by severe prosopagnosia, semantic impairment, and behavioral modifications. In these three patients, the molecular analysis of MAPT showed the c.1945C>T mutation on exon 11 resulting in the P332S substitution in tau sequence. This mutation changes the PGGG motif of the third repeat domain of the protein and therefore reduces the ability of tau to bind microtubule. From a clinical point of view, this mutation is associated with considerable intrafamilial phenotypic variation.

Clinical, neuropathological, and biochemical characterization of the novel tau mutation P332S.

The τ pathology found in Alzheimer disease (AD) is crucial in cognitive decline. Midlife development of obesity, a major risk factor of insulin resistance and type 2 diabetes, increases the risk of dementia and AD later in life. The impact of obesity on AD risk has been suggested to be related to central insulin resistance, secondary to peripheral insulin resistance. The effects of diet-induced obesity (DIO) on τ pathology remain unknown. In this study, we evaluated effects of a high-fat diet, given at an early pathological stage, in the THY-Tau22 transgenic mouse model of progressive AD-like τ pathology. We found that early and progressive obesity potentiated spatial learning deficits as well as hippocampal τ pathology at a later stage. Surprisingly, THY-Tau22 mice did not exhibit peripheral insulin resistance. Further, pathological worsening occurred while hippocampal insulin signaling was upregulated. Together, our data demonstrate that DIO worsens τ phosphorylation and learning abilities in τ transgenic mice independently from peripheral/central insulin resistance.

Detrimental effects of diet-induced obesity on τ pathology are independent of insulin resistance in τ transgenic mice.

Myotonic dystrophy (DM) of type 1 and 2 (DM1 and DM2) are inherited autosomal dominant diseases caused by dynamic and unstable expanded microsatellite sequences (CTG and CCTG, respectively) in the non-coding regions of the genes DMPK and ZNF9, respectively. These mutations result in the intranuclear accumulation of mutated transcripts and the mis-splicing of numerous transcripts. This so-called RNA gain of toxic function is the main feature of an emerging group of pathologies known as RNAopathies. Interestingly, in addition to these RNA inclusions, called foci, the presence of neurofibrillary tangles (NFT) in patient brains also distinguishes DM as a tauopathy. Tauopathies are a group of nearly 30 neurodegenerative diseases that are characterized by intraneuronal protein aggregates of the microtubule-associated protein Tau (MAPT) in patient brains. Furthermore, a number of neurodegenerative diseases involve the dysregulation of splicing regulating factors and have been characterized as spliceopathies. Thus, myotonic dystrophies are pathologies resulting from the interplay among RNAopathy, spliceopathy, and tauopathy. This review will describe how these processes contribute to neurodegeneration. We will first focus on the tauopathy associated with DM1, including clinical symptoms, brain histology, and molecular mechanisms. We will also discuss the features of DM1 that are shared by other tauopathies and, consequently, might participate in the development of a tauopathy. Moreover, we will discuss the determinants common to both RNAopathies and spliceopathies that could interfere with tau-related neurodegeneration.

Francisco-Jose Fernandez-Gomez

Team Alzheimer & Tauopathies,
Jean-Pierre Aubert Research Centre,
Team Alzheimer & Tauopathies, Jean-Pierre Aubert Research Centre

Inserm UMR 837

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Francisco-Jose Fernandez-Gomez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Team Alzheimer & Tauopathies,Jean-Pierre Aubert Research Centre,Team Alzheimer & Tauopathies, Jean-Pierre Aubert Research Centre

Inserm UMR 837

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Team Alzheimer & Tauopathies,
Jean-Pierre Aubert Research Centre,
Team Alzheimer & Tauopathies, Jean-Pierre Aubert Research Centre