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1.
Animals (Basel) ; 13(14)2023 Jul 12.
Article in English | MEDLINE | ID: mdl-37508055

ABSTRACT

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

2.
Emerg Infect Dis ; 26(1): 34-43, 2020 01.
Article in English | MEDLINE | ID: mdl-31855141

ABSTRACT

Variant Creutzfeldt-Jakob disease (vCJD) is caused by prion infection with bovine spongiform encephalopathy and can be transmitted by blood transfusion. Protein misfolding cyclic amplification (PMCA) can detect prions in blood from vCJD patients with 100% sensitivity and specificity. To determine whether PMCA enables prion detection in blood during the preclinical stage of infection, we performed a blind study using blood samples longitudinally collected from 28 control macaques and 3 macaques peripherally infected with vCJD. Our results demonstrate that PMCA consistently detected prions in blood during the entire preclinical stage in all infected macaques, without false positives from noninfected animals, when using the optimized conditions for amplification of macaque prions. Strikingly, prions were detected as early as 2 months postinoculation (>750 days before disease onset). These findings suggest that PMCA has the potential to detect vCJD prions in blood from asymptomatic carriers during the preclinical phase of the disease.


Subject(s)
Creutzfeldt-Jakob Syndrome/veterinary , Prions/blood , Animals , Blotting, Western , Creutzfeldt-Jakob Syndrome/blood , Creutzfeldt-Jakob Syndrome/diagnosis , Macaca , Prodromal Symptoms , Reproducibility of Results , Sensitivity and Specificity
3.
Proc Natl Acad Sci U S A ; 112(7): 2239-44, 2015 Feb 17.
Article in English | MEDLINE | ID: mdl-25646456

ABSTRACT

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.


Subject(s)
Corpus Striatum/physiopathology , Disease Models, Animal , Homeostasis , Huntington Disease/physiopathology , Locomotion , Animals , Mice
4.
Mol Cell Neurosci ; 52: 117-27, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23147111

ABSTRACT

Proteins of the major histocompatibility complex class I (MHCI) are known for their role in the vertebrate adaptive immune response, and are required for normal postnatal brain development and plasticity. However, it remains unknown if MHCI proteins are present in the mammalian brain before birth. Here, we show that MHCI proteins are widely expressed in the developing mouse central nervous system at mid-gestation (E9.5-10.5). MHCI is strongly expressed in several regions of the prenatal brain, including the neuroepithelium and olfactory placode. MHCI is expressed by neural progenitors at these ages, as identified by co-expression in cells positive for neuron-specific class III ß-tubulin (Tuj1) or for Pax6, a marker of neural progenitors in the dorsal neuroepithelium. MHCI is also co-expressed with nestin, a marker of neural stem/progenitor cells, in olfactory placode, but the co-localization is less extensive in other regions. MHCI is detected in the small population of post-mitotic neurons that are present at this early stage of brain development, as identified by co-expression in cells positive for neuronal microtubule-associated protein-2 (MAP2). Thus MHCI protein is expressed during the earliest stages of neuronal differentiation in the mammalian brain. MHCI expression in neurons and neural progenitors at mid-gestation, prior to the maturation of the adaptive immune system, is consistent with MHCI performing non-immune functions in prenatal brain development. These results raise the possibility that disruption of the levels and/or patterns of MHCI expression in the prenatal brain could contribute to the pathogenesis of neurodevelopmental disorders.


Subject(s)
Brain/embryology , Brain/metabolism , Histocompatibility Antigens Class I/metabolism , Neural Stem Cells/metabolism , Neurons/metabolism , Animals , Fluorescent Antibody Technique , Immunohistochemistry , Mice , Mice, Inbred C57BL , Neural Stem Cells/cytology , Neurons/cytology
5.
J Cell Physiol ; 217(1): 215-27, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18521822

ABSTRACT

The activation of the canonical Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimer's amyloid-beta-peptide (Abeta), however, the role played by the Wnt receptors Frizzleds, has not been studied. We report here that Frizzled-1 mediates the activation of the canonical Wnt/beta-catenin pathway by Wnt3a in PC12 cells. In addition, the protective effect of Wnt3a against the toxicity of Abeta oligomers was modulated by Frizzled-1 expression levels in both PC12 cells and hippocampal neurons. Over-expression of Frizzled-1 significantly increased cell survival induced by Wnt3a and diminished caspase-3 activation, while knocking-down Frizzled-1 expression by antisense oligonucleotides decreased the Wnt3a protection. Over-expression of wild-type beta-catenin, but not a transcriptionally inactive mutated version, prevented the toxicity of Abeta suggesting that the transcription of Wnt target genes may be involved in these events. This was confirmed by co-transfecting both Frizzled-1 and the inactive form of beta-catenin, which does not elicited protection levels similar to those showed with endogenous beta-catenin. Our results indicate that Wnt3a protects from Abeta-oligomers toxicity by activating the canonical Wnt signaling pathway through the Frizzled-1 receptor, suggesting a therapeutic potential for this signaling pathway in the treatment of Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides/toxicity , Frizzled Receptors/metabolism , Nerve Degeneration/metabolism , Signal Transduction/physiology , Wnt Proteins/metabolism , Animals , Blotting, Western , Caspase 3/metabolism , Cell Line , Cell Survival/physiology , Cells, Cultured , Fluorescent Antibody Technique , Hippocampus/metabolism , Hippocampus/pathology , Immunohistochemistry , Neurons/metabolism , Neurons/pathology , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Wnt3 Protein , beta Catenin/metabolism
6.
Biol Res ; 39(1): 39-44, 2006.
Article in English | MEDLINE | ID: mdl-16629163

ABSTRACT

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.


Subject(s)
Copper/metabolism , Oxidative Stress/physiology , PrPC Proteins/metabolism , Animals , Heparan Sulfate Proteoglycans/metabolism , PrPC Proteins/genetics , Prion Diseases/metabolism , Protein Binding , Rats
7.
Biol. Res ; 39(1): 39-44, 2006. tab
Article in English | LILACS | ID: lil-430696

ABSTRACT

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.


Subject(s)
Animals , Rats , Copper/metabolism , Oxidative Stress/physiology , PrPC Proteins/metabolism , Heparan Sulfate Proteoglycans/metabolism , Protein Binding , PrPC Proteins/genetics , Prion Diseases/metabolism
8.
Neurobiol Aging ; 26(7): 1023-8, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15748782

ABSTRACT

It is generally accepted that human Alzheimer's disease (AD) neuropathology markers are completely absent in rodent brains. We report here that an aged wild-type South American rodent, Octodon degu, expresses neuronal beta-amyloid precursor protein (beta-APP695) displaying both intracellular and extracellular deposits of amyloid-beta-peptide (Abeta), intracellular accumulations of tau-protein and ubiquitin, a strong astrocytic response and acetylcholinesterase (AChE)-rich pyramidal neurons. The high amino acid homology (97.5%) between deguAbeta and humanAbeta sequences is probably a major factor in the appearance of AD markers in this aged rodent. Our results indicate that aged O. degu constitutes the first wild-type rodent model for neurodegenerative processes associated to AD.


Subject(s)
Aging/physiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Octodon/metabolism , Acetylcholinesterase/metabolism , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid beta-Peptides/genetics , Animals , Astrocytes/metabolism , Blotting, Northern/methods , Brain/cytology , Brain/metabolism , Disease Models, Animal , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Humans , Immunohistochemistry/methods , Neurons/metabolism , RNA, Messenger/biosynthesis , Rats , Reverse Transcriptase Polymerase Chain Reaction/methods , Ubiquitin/metabolism , tau Proteins/metabolism
9.
FASEB J ; 18(14): 1701-3, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15345692

ABSTRACT

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135-156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra-hippocampal injection model. We found that human APP135-156 protects against Cu2+-induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135-156 lacking two histidine residues showed protection against Cu2+; however, APP135-156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL-1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/physiology , Copper/metabolism , Copper/toxicity , Tyrosine/analogs & derivatives , Amyloid beta-Protein Precursor/therapeutic use , Animals , Binding Sites , Caenorhabditis elegans Proteins/chemistry , Copper/antagonists & inhibitors , Cysteine/chemistry , Gliosis/chemically induced , Gliosis/prevention & control , Humans , Ion Transport , Membrane Proteins/chemistry , Memory/drug effects , Neurons/cytology , Neurotoxicity Syndromes/prevention & control , Peptides/chemistry , Peptides/therapeutic use , Protein Structure, Tertiary , Proteins/chemistry , Rats , Tyrosine/analysis
10.
Am J Pathol ; 164(6): 2163-74, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15161650

ABSTRACT

Neuropathological changes generated by human amyloid-beta peptide (Abeta) fibrils and Abeta-acetylcholinesterase (Abeta-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Abeta-AChE complexes trigger a more dramatic response in situ than Abeta fibrils alone as characterized by the following features observed 8 weeks after treatment: 1). amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Abeta aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Abeta-AChE deposits observed in vivo probably recruited endogenous Abeta peptide; 2). the appearance of laminin expressing neurons surrounding Abeta-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3). an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4). a stronger neuronal cell loss in comparison with Abeta-injected animals. We conclude that the hippocampal injection of Abeta-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Abeta-AChE complexes are more toxic than Abeta fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimer's disease brains.


Subject(s)
Acetylcholinesterase/toxicity , Amyloid beta-Protein Precursor/toxicity , Astrocytes/pathology , Gliosis/pathology , Hippocampus/pathology , Laminin/genetics , Neurons/pathology , Animals , Astrocytes/drug effects , Cattle , Cell Survival/drug effects , Glial Fibrillary Acidic Protein/analysis , Gliosis/chemically induced , Hippocampus/drug effects , Humans , Image Processing, Computer-Assisted , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Spectrometry, Fluorescence
11.
J Neurochem ; 87(1): 195-204, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12969266

ABSTRACT

Previous studies have demonstrated that acetylcholinesterase (AChE) promotes the assembly of amyloid-beta-peptides into neurotoxic amyloid fibrils and is toxic for chick retina neuronal cultures and neuroblastoma cells. Moreover, AChE is present in senile plaques in Alzheimer's disease (AD) brains. Here we have studied the effect of AChE on astrocytes and hippocampal neurons in vivo. Morphological as well as behavioral disturbances were analyzed after intrahippocampal injection of AChE. Rats were trained in the Morris water maze and assayed for behavioral parameters. Neuronal cell loss was found in the upper leaf of the dentate gyrus in rats injected with AChE in comparison with control animals. Glial fibrillary acidic protein immunoreactivity showed astrocytic hypertrophy and the magnitude of the response was associated with neuronal cell loss. Behavioral results show that injection of AChE produces cognitive impairment demonstrated by an altered water maze performance including (i) a higher escape latency score, (ii) a decreased spatial acuity and (iii) a shorter time of swimming in the platform quadrant. These findings indicate that a local increment in neuronal AChE concentration at the mammalian hippocampus, such as those present in amyloid deposits, may play a role in triggering neuropathological and behavioral changes such as those observed in AD brains.


Subject(s)
Acetylcholinesterase/pharmacology , Astrocytes/drug effects , Hippocampus/drug effects , Hippocampus/physiopathology , Neurons/drug effects , Animals , Astrocytes/metabolism , Astrocytes/pathology , Behavior, Animal/drug effects , Cattle , Drug Administration Routes , Glial Fibrillary Acidic Protein/biosynthesis , Hippocampus/pathology , Male , Maze Learning/drug effects , Neurons/pathology , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects
12.
Neurochem Int ; 41(5): 341-4, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12176076

ABSTRACT

Alzheimer's disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-beta peptide (Abeta). We have observed that Abeta-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer's patients.


Subject(s)
Amyloid beta-Peptides/physiology , Nerve Degeneration/physiopathology , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , Zebrafish Proteins , Animals , Behavior, Animal/physiology , Humans , Rats , Wnt Proteins
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