Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus).

Coagulation factor X (FX)-binding ablated adenovirus type 5 (Ad5) vectors have been genetically engineered to ablate the interaction with FX, resulting in substantially reduced hepatocyte transduction following intravenous administration in rodents. Here, we quantify viral genomes and gene transfer mediated by Ad5 and FX-binding-ablated Ad5 vectors in non-human primates. Ad5 vectors accumulated in and mediated gene transfer predominantly to the liver, whereas FX-binding-ablated vectors primarily targeted the spleen but showed negligible liver gene transfer. In addition, we show that Ad5 binding to hepatocytes may be due to the presence of heparan sulfate proteoglycans (HSPGs) on the cell membrane. Therefore, the Ad5-FX-HSPG pathway mediating liver gene transfer in rodents is also the mechanism underlying Ad5 hepatocyte transduction in Microcebus murinus.

The grey mouse lemur: a non-human primate model for ageing studies.

The use of non-human primate models is required to understand the ageing process and evaluate new therapies against age-associated pathologies. The present article summarizes all the contributions of the grey mouse lemur Microcebus murinus, a small nocturnal prosimian primate, to the understanding of the mechanisms of ageing. Results from studies of both healthy and pathological ageing research on the grey mouse lemur demonstrated that this animal is a unique model to study age-dependent changes in endocrine systems, biological rhythms, thermoregulation, sensorial, cerebral and cognitive functions.

Immunohistochemical analysis of cerebral cortical and vascular lesions in the primate Microcebus murinus reveal distinct amyloid beta1-42 and beta1-40 immunoreactivity profiles.

Recent reports have shown that amyloid beta deposits in the brains of Alzheimer's disease patients consist mainly of two distinct species of amyloid beta protein (Abeta) with different C-termini, Abeta1-42 (Abeta42) and Abeta1-40 (Abeta40). The nature of the Abeta species in Microcebus murinus brain was investigated immunocytochemically using polyclonal antibodies with clear specificity for the Abeta42 and Abeta40 C-termini. The cortical vascular deposits were immunopositive for both Abeta42 and Abeta40. However, most of the diffuse plaques were strongly positive for Abeta42 whereas only a subset of deposits were positive for Abeta40. Numerous cortical plaques were Abeta42-immunopositive but tested negative for Abeta40. This suggests that Abeta42 is probably associated with early stages of plaque maturation. This neuropathological feature reminiscent of that observed in brains affected by Alzheimer's disease further supports the idea that M. murinus could be used as a potential model of the early stages of this neurological disease.

Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents.

Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.

A stereotaxic atlas of the grey lesser mouse lemur brain (Microcebus murinus).

In response to the growing interest in the prosimian Microcebus murinus for studies on cerebral aging, the stereotaxic atlas of its brain was carried out in view of further anatomical, biochemical, electrophysiological, and behavioral investigations as well as for therapeutic experiments. This primate, which could be a valuable model for neuroscientific studies in various domains, presents numerous physiological advantages (e.g., size, cost, and ability to breed) compared to rodents, which can be used as nonprimate models, and simians. The atlas, valid for adult microcebes of every age and both sexes, consists of 54 frontal plates and 28 sagittal plates. For the establishment of stereotaxic coordinates and for drawings and photographs, 10 adult specimens of Microcebus murinus were used. The brains were frozen, cut into sections of 50 microm thickness, every fourth section being stained with Nissl. First, sections were projected and the outlines of the different structures, nuclei, and fibers were drawn. Then, the accuracy of the analysis was improved by detailed observation directly by microscope and also by computer analysis. Finally, the photographs of the sections were scanned and processed using the software Photoshop and Illustrator. For testing coordinates, several verifications were made. Experiments on lesions and injections of different substances were carried out in specific regions of the brain and brains implanted with needles were fixed in formol and embedded in paraffin wax.

Cloning of the presenilin 2 cDNA and its distribution in brain of the primate, Microcebus murinus: coexpression with betaAPP and Tau proteins.

A 1340-bp cDNA fragment encoding the lemurian presenilin 2 protein (PS2) was isolated from a Microcebus murinus brain cDNA library by PCR using oligonucleotide primers based on the nucleotide sequence of the human gene. Analysis of five isolated clones showed that the sequence encoded a 448-amino-acid open reading frame, 95.5% identical to the human and 93.5% identical to the mouse presenilin 2 sequences. However, neither the localization of the 2 positions in PS2 nor that of the 43 positions in PS1 associated with early onset Alzheimer's disease were changed. Expression of the presenilin 2 was detected by RT-PCR and compared with that of presenilin 1 and betaAPP in the brain and in peripheral tissues (liver, kidney, and spleen). Immunohistochemistry with a specific polyclonal antiserum raised against a synthetic peptide from the N-terminal part of the human PS2 showed that the protein is distributed throughout the microcebe brain, in vascular and nerve structures. In cortical and in subcortical areas, PS2 labeling was weak and granular in appearance and was scattered throughout the cytoplasm of many neurones, extending into neurites. The gene expression of PS2 increased with age but was not affected by the presence of numerous amyloid plaques. Double labeling immunocytochemistry detected very few neurones with combined immunoreactivity PS2 and APP, or PS2 and Tau.

Prion immunoreactivity in brain, tonsil, gastrointestinal epithelial cells, and blood and lymph vessels in lemurian zoo primates with spongiform encephalopathy.

We report on two animals of a non-human primate species Eulemur fulvus mayottensis, housed in the local zoo and fed over a number of years with a food containing cattle meat, that developed serious neurological symptoms associated with prion immunoreactivity in brain and various viscera. Microscopy of the brains showed neuronal vacuolation with patchy/perivacuolar immunolabelling with an abnormal isoform of prion protein (IR-PrP), an important characteristic of spongiform encephalopathy. For the first time, we report the presence in the same severely ill animals of IR-PrP in the gastrointestinal tract, detected by immunocytochemistry with mono- and polyclonal antibodies directed against various parts of the PrP. Strong PrP labelling was observed in the epithelial cells lining the pharyngeal and gastrointestinal lumen. The tonsils and the walls of the lymph and blood vessels below the intestinal epithelium were also labelled. There were no such immunoreactions in healthy lemurians killed as controls, i.e. a younger congener of the same species housed under the same conditions, and others belonging to the smaller species Microcebus murinus, reared in the laboratory and never fed on commercial food products containing cattle meat. These results demonstrate a strong PrP accumulation in the brain, the gastrointestinal tract and underlying lymphoreticular structures in these primates living in a zoological park and suffering from a spongiform encephalopathy.

Molecular cloning, sequencing, and brain expression of the presenilin 1 gene in Microcebus murinus.

The cDNA encoding the Microcebus murinus presenilin 1 protein (PSI) was cloned by RT-PCR from a brain cDNA library using various combinations of oligonucleotide primers designed on the basis of the human nucleotide sequence. Analysis of five clones isolated from two positive combinations revealed that the deduced open reading frame encodes two protein isoforms of 467 and 463 amino acid residues. The shorter isoform lacked the four residues VRSQ in the N-terminal region and like the 467 amino acid isoform presented 22 substitutions with its human homologue. The 12 bp nucleotide deletion evidenced in the cDNA encoding the shorter isoform is consistent with the use of an alternative 5' splice donor site identified at the end of the human exon 3. The immunohistochemistry performed with a specific polyclonal antiserum raised against a synthetic peptide located in the human large hydrophilic loop of PS1 revealed that the protein is widely distributed independently of age or of pathology in the microcebe brain. PS1 is found predominantly in neurons of the different cortical layers and hippocampus but also in subcortical structures. The PS1 labelling appeared as thin granulations scattered throughout the cytoplasm of numerous neurons and sometimes in neurites.

Spontaneous spongiform encephalopathy in a young adult rhesus monkey.

This paper reports the occurrence of a spongiform encephalopathy (SE) in young adult monkeys housed in a zoological park. Three rhesus monkeys (Macaca mulatta) acquired from the same zoo and maintained on feed containing animal protein, developed a progressive neurological disorder with behavioural abnormalities and physical deterioration and died at the age of 10-year-old. Neuropathological examination of one of these animals revealed a spongiform encephalopathy similar to that observed in monkeys following experimental transmission of Creutzfeldt-Jakob disease (CJD). In particular, several brain regions exhibited vacuolation of nerve cell bodies and processes accompanied by astrogliosis. Immunohistochemical analysis showed prion protein (PrP) immunoreactivity at the periphery of vacuolated neurons. The spontaneous occurrence of a SE in these young monkeys might be related to consumption of protein of animal origin.

beta-Amyloid protein precursor in Microcebus murinus: genotyping and brain localization.

Senile plaques characterized by beta-amyloid protein (A beta) deposits around dystrophic neurites and glial cells are more abundant in the cerebral cortical parenchyma of Alzheimer's disease (AD) patients than in the aged population. Four different mutations in the amyloid precursor protein (APP) gene have been directly involved in a few cases of familial AD with early onset (before 60 years). Previous studies have shown that Microcebus murinus, a nonhuman primate, also develops analogous deposits of A beta in the cortical parenchyma and blood vessel walls in the brain. Sequence analysis of exons 16 and 17 of the APP gene, encoding for A beta, revealed that even if nucleotide divergences occurred, the resulting peptide is completely homologous with the human A beta. The systematic comparison of the A beta nucleotide sequence in microcebus with or without amyloid deposits revealed that neither the presence of mutations involved in some cases of early onset familial AD nor the presence of a mutational founder effect can explain the amyloidosis observed in some old microcebus of our breeding. Localization of the APP was performed by immunocytochemistry in the brains of adult microcebus (1 to 11 years of age) using two antibodies raised against the C-terminus and N-terminus portions of APP. Microscopic examinations revealed that in the microcebus the APP distribution was similar to that observed in the human: (1) A beta and its precursor were simultaneously observed in amyloid plaques (AP) of the cortical parenchyma; (2) APP was localized in cell bodies and proximal dendrites of neurons, in astrocytes and oligodendrocytes, and in blood vessel and capillary walls; (3) labeling of APP in these structures was correlated with the presence of AP; and (4) labeling of APP increased with the age of the animal.

Immunocytochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus.

The immunocytochemistry of Tau proteins in the cortical pyramidal neurons of the adult microcebes has been studied, using antibodies against human normal and pathological Tau proteins. Some changes related to the age and to some pathologies were observed. In fact, during the adult life, Tau proteins appeared as very thin granulations scattered in the whole neuronal cytoplasm. With age, a part of these proteins aggregated and became like thick granules at the neuron periphery; the distribution was not uniform, and numerous neurons with aggregated Tau proteins were observed in amyloid plaque-containing brains. Abnormally phosphorylated Tau proteins were also observed in some aged animals, using an absorbed anti-PHF recognizing the pathological Tau proteins characteristic of Alzheimer's disease. This present work confirms that the microcebe is a good model for studying disfunctions involved in the normal cerebral aging and in some neurodegenerative disorders which affect humans.

Immunocytochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus.

The immunocytochemistry of Tau proteins in the cortical pyramidal neurons of the adult microcebes has been studied, using antibodies against human normal and pathological Tau proteins. Some changes related to the age and to some pathologies were observed. In fact, during the adult life, Tau proteins appeared as very thin granulations scattered in the whole neuronal cytoplasm. With age, a part of these proteins aggregated and became like thick granules at the neuron periphery; the distribution was not uniform, and numerous neurons with aggregated Tau proteins were observed in amyloid plaque-containing brains. Abnormally phosphorylated Tau proteins were also observed in some aged animals, using an absorbed anti-PHF recognizing the pathological Tau proteins characteristic of Alzheimer's disease. This present work confirms that the microcebe is a good model for studying disfunctions involved in the normal cerebral aging and in some neurodegenerative disorders which affect humans.