It has been suggested that systemic infection, occurring during aging and chronic neurodegenerative diseases, can evoke an immune response that aggravates the progression of neurodegeneration and ...cognitive decline. It has been shown that the AD11 neurodegeneration mouse model, expressing a recombinant anti-nerve growth factor (NGF) antibody, shows a milder phenotype when housed in murine pathogen-free (MPF) conditions with respect to AD11 mice reared in conventional (CV) housing. AD10 mice, a variant of the anti-NGF AD11 model, expressing only an immunoglobulin light chain for the transgenic anti-NGF antibody, in the absence of the corresponding transgenic antibody chain VH, exhibit a complex neurodegenerative phenotype, similar to that of AD11 mice. Here we show that the AD10 transgenic mice, housed in murine pathogen-free conditions (MPF-AD10 mice), also display a milder behavioral and neurodegenerative phenotype compared to the corresponding mice kept under conventional housing conditions (CV-AD10). As a first step toward the identification of mechanisms underlying this difference, a differential gene expression profiling was performed on brains from CV-AD10 and MPF-AD10 mice, showing a decrease of the immune response and neuroinflammation gene expression in MPF-AD10 mice. Results suggest that the activation of the immune response gene expression in the CV-AD10, in a microbially unprotected environment, might contribute to a more severe and progressive neurodegenerative phenotype, compared to the MPF mice.
Metabotropic glutamate (mGlu) receptors have been considered as potential targets for neuroprotective drugs, but the lack of specific drugs has limited the development of neuroprotective strategies ...in experimental models of acute or chronic central nervous system (CNS) disorders. The advent of potent and centrally available subtype-selective ligands has overcome this limitation, leading to an extensive investigation of the role of mGlu receptor subtypes in neurodegeneration during the last 2 years. Examples of these drugs are the noncompetitive mGlu1 receptor antagonists, CPCCOEt and BAY-36-7620; the noncompetitive mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)pyridine, SIB-1893, and SIB-1757; and the potent mGlu2/3 receptor agonists, LY354740 and LY379268. Pharmacologic blockade of mGlu1 or mGlu5 receptors or pharmacologic activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of N-methyl-d-aspartate (NMDA) receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes. A series of observations suggest a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer disease. MGlu2/3 receptor agonists inhibit glutamate release, but also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of CNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders. The advantage of all these drugs with respect to NMDA or AMPA receptor agonists derives from the evidence that mGlu receptors do not “mediate,” but rather “modulate” excitatory synaptic transmission. Therefore, it can be expected that mGlu receptor ligands are devoid of the undesirable effects resulting from the inhibition of excitatory synaptic transmission, such as sedation or an impairment of learning and memory.
Background
The quantification of cerebrospinal fluid (CSF) biomarkers (Abeta peptides Ab1‐40 and Ab1‐42, tau protein and its phosphorylated form phospho‐tau) is progressively implemented in ...laboratories as an aid for the multidisciplinary diagnosis of Alzheimer disease (AD), DeKosky ST, Alz dementia, 2011, PMID: 21322828 . However, no consensus has been defined among the different laboratories involved to adapt the conclusions/comments to the level of quantified CSF biomarkers. As a result, although the analytical methods for such quantification may be similar across the laboratories involved in this clinical task, the conclusions transmitted to the physician in charge (neurologist or psychiatrist) may be quite different. Harmonization of this report is thus necessary so patients’ care and research stratification can be similar wherever the analysis is performed.
Method
A total of 34 laboratories (involved in CSF biomarkers measurement) across the world accepted to be part of our project of diagnostic’s comments harmonization (represented countries: Austria, Belgium, Canada, France, Germany, Italy, Netherland, Poland, Spain, Sweden, United Kingdom, USA). As a first step, we defined the 9 most typical biochemical profiles, according to the level of CSF biomarkers and their combination. For each profile, each laboratory was asked to provide us with the comments/conclusions given in routine clinical practice. We then collected and pooled all the comments in a common file, so that the laboratories could, as a second step, choose and order three of these comments (for each biochemical profile defined), according to their reliability in clinical practice.
Result
We are currently analysing the second step‐answers of the laboratories, in order to define a consensual pattern of comments and conclusions that could be implemented in all the laboratories involved in the biochemical diagnosis of AD. Obtained data will be presented.
Conclusion
The discrepancies of the comments for AD biochemical diagnosis across laboratories worldwide can be confusing and it is of strong importance to harmonize them (according to the level of quantified biomarkers and other information likely available such as the age, APOE genotype...). Our initiative will likely provide such harmonized pattern of comments/conclusions, thus ensuring equal care of patients across the different diagnostic centres.
Abstract
Background
The quantification of cerebrospinal fluid (CSF) biomarkers (Abeta peptides Ab
1‐40
and Ab
1‐42
, tau protein and its phosphorylated form phospho‐tau) is progressively implemented ...in laboratories as an aid for the multidisciplinary diagnosis of Alzheimer disease (AD),
DeKosky ST, Alz dementia, 2011, PMID: 21322828
. However, no consensus has been defined among the different laboratories involved to adapt the conclusions/comments to the level of quantified CSF biomarkers. As a result, although the analytical methods for such quantification may be similar across the laboratories involved in this clinical task, the conclusions transmitted to the physician in charge (neurologist or psychiatrist) may be quite different. Harmonization of this report is thus necessary so patients’ care and research stratification can be similar wherever the analysis is performed.
Method
A total of 34 laboratories (involved in CSF biomarkers measurement) across the world accepted to be part of our project of diagnostic’s comments harmonization (represented countries: Austria, Belgium, Canada, France, Germany, Italy, Netherland, Poland, Spain, Sweden, United Kingdom, USA). As a first step, we defined the 9 most typical biochemical profiles, according to the level of CSF biomarkers and their combination. For each profile, each laboratory was asked to provide us with the comments/conclusions given in routine clinical practice. We then collected and pooled all the comments in a common file, so that the laboratories could, as a second step, choose and order three of these comments (for each biochemical profile defined), according to their reliability in clinical practice.
Result
We are currently analysing the second step‐answers of the laboratories, in order to define a consensual pattern of comments and conclusions that could be implemented in all the laboratories involved in the biochemical diagnosis of AD. Obtained data will be presented.
Conclusion
The discrepancies of the comments for AD biochemical diagnosis across laboratories worldwide can be confusing and it is of strong importance to harmonize them (according to the level of quantified biomarkers and other information likely available such as the age, APOE genotype...). Our initiative will likely provide such harmonized pattern of comments/conclusions, thus ensuring equal care of patients across the different diagnostic centres.
We examined the expression of metabotropic glutamate (mGlu) receptors in species of fish that differ for their vulnerability to anoxic brain damage. Although expression of mGlu1a and mGlu5 receptors ...was similar in the brain of all species examined, expression of mGlu2/3 receptors was substantially higher in the brain of anoxia-tolerant species (i.e., the carp Carassius carassius and the goldfish Carassius auratus) than in the brain of species that are highly vulnerable to anoxic damage, such as the trouts Salmo trutta and Oncorhynchus mykiss. This difference was confirmed by measuring the mGlu2/3 receptor-mediated inhibition of forskolin-stimulated cAMP formation in slices prepared from the telencephalon of C. auratus and S. trutta. We exposed the goldfish C. auratus to water deprived of oxygen for 4 hr for the induction of hypoxic brain damage. Although the goldfish survived this treatment, the occurrence of apoptotic cell death could be demonstrated by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining and by the assessment of caspase-3 activity in different brain region. The extent of cell death was highest in the medulla oblongata, followed by the optic tectum, cerebellum, and hypothalamus. No cell death was found in the telencephalon. This regional pattern of hypoxic damage was inversely related to the expression of mGlu2/3 receptors, which was lowest in the medulla oblongata and highest in the telencephalon. Treatment of the goldfish with the brain permeant mGlu2/3 receptor antagonist LY341495 (1 mg/kg, i.p.) amplified anoxic damage throughout the brain and enabled the induction of cell death by anoxia in the telencephalon. In contrast, treatment of the goldfish with the mGlu2/3 receptor agonist LY379268 (0.5 or 1 mg/kg, i.p.) was highly protective against anoxic brain damage. Finally, exposure to the antagonist LY341495 (0.5 microm) greatly amplified the release of glutamate induced by hypoxia in slices prepared from the medulla oblongata and the telencephalon of the goldfish. We conclude that expression of mGlu2/3 receptors provides a major defensive mechanism against brain damage in anoxia-tolerant species.
The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine ...the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a λ gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a β -galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.
Glycoproteins of the nuclear pore complex are thought to play an important role in the transport of regulatory proteins and
ribonucleoproteins across the nuclear envelope. However, the genetic ...elements and signals that control the expression of nuclear
pore glycoproteins are poorly understood. To study the transcriptional regulation of mammalian nuclear pore glycoprotein biosynthesis,
we have isolated the gene coding for the major rat nuclear pore glycoprotein p62. The p62 gene consists of a 2941-base pair
region that is linear with the full length p62 cDNA with no intervening sequences. Quantitative Southern analysis revealed
that the gene is present in single copy. The p62 gene encodes a 525-amino acid open reading frame that directs the synthesis
of the 62-kDa pore glycoprotein in vitro and in transfected cultured cells. The 5'-flanking region contains two potential
transcription start sites; primer extension analysis revealed that the furthest upstream site is preferentially used in vivo.
When linked to a reporter gene, the 5'-flanking region of the p62 gene serves as an active promoter.
The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na
+/K
+-ATPase has been investigated. When native Na
+/K
+-ATPase is exposed to ...guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na
+/K
+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na
+/K
+-ATPase (ouabain) (
K
SV = 15.6 M
−1) with respect to the native enzyme (
K
SV = 12.5 M
−1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na
+/K
+-ATPase(ouabain) (
K
SV = 4 M
−1) compared to the Na
+/K
+-ATPase (
K
SV = 7 M
−1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent, These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na
+/K
+-ATPase conformation.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are ...associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression.
SYNOPSIS
A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis.
MaMTH‐HTS identifies 447 interactors of wildtype and F508del CFTR.
The CFTR interactomes are evaluated using traditional MaMTH and a fluorescence‐based assay is performed to monitor the effect of transiently expressed interactors on CFTR channel activity.
siRNA‐mediated knockdown of candidate proteins reveals 19 interactors whose down‐regulation led to increased F508del CFTR trafficking and complex glycosylation.
One candidate protein, Fibrinogen Like 2 (FGL2) has a significant effect on CFTR functional expression, as demonstrated in patient‐derived intestinal organoids.
A new MaMTH‐HTS platform is used with a Human ORFeome library to map the protein‐protein interactions of full‐length wildtype and F508del CFTR. Functional validations in multiple disease models uncovered proteins with potential roles in CFTR function and cystic fibrosis.
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