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See Also: 1.1, 1.6, 1.7, 1.8, 8.0, 8.1

2.0 Co-expression of Presenilin and mutated Amyloid Precursor Protein in transgenic mice. C Czech*, S Dreisler, N Touchet, N Clavel, B Schombert, N Buron, G Ret, L Pradier, and G.L Tremp. Rhône-Poulenc Rorer S.A., GENE & MEDICINE Department, Centre de Recherche Vitry-Alfortville, 94403 Vitry sur Seine, France. E-mail: Christian.Czech@rhone-poulenc.com

Keywords: Presenilin, Amyloid Precursor Protein, Transgenic Mice

Abstract: Both environmental and genetic factors are involved in AD aetiology. Mutations in APP and in PS1 and PS2 genes are causing early-onset forms of the disease while the ApoEe4 allele is a risk factor for AD. Furthermore, overexpression of mutated forms of APP in transgenic animals has now been shown to lead to amyloid plaque formation. However, rather than large overexpression, transgenic models based on a combination of the known genetic factors expressed at more physiological levels might lead to a better understanding of the pathology. Additional external stress factors like trauma or lesions could further contribute to the development of AD in these animals. Towards that goal, we have generated a human triple mutant APP (SDL) transgenic mouse with moderate expression levels of the transgene. Ab peptide production is clearly detected in these animals, but without formation of amyloid plaques. In addition wild-type PS1 transgenic mice and rats and mutated PS1 mice have also been generated. The transgenic PS1 protein is subject to proteolytical processing in the brain leading to the two characteristic N- and C-terminal fragments which can be clearly differentiated by size from the endogenous mouse but not rat fragments. That processing is saturable in the high expressing lines with apparition of the full-length (51 kD) protein and is not affected by the mutation M146L. We have not been able to identify AD related pathology in these mice. To study the interaction between PS 1 and APP in vivo, we created mice expressing both, mutated human APP and human PS 1 with and without the FAD mutation M146L. Analysis of APP processing in double transgenic mice (mut APP and wt PS-1 or mut APP PS-1 M146L) did not reveal significant changes in total Aß levels however a possible increase of Ab 1-42 remains to be elucidated. Furthermore the processing of either wild-type or mutated PS-1 in the double trangenics remained unchanged. Immunohistological analysis of double transgenic mice is in progress. Since AD is a multigenic disease, we combined several AD linked genes and risk factors in the same animal. A better understanding of the genetic and extra-genetic events; their interaction and timing, leading to AD is crucial for the development of an innovative therapeutic approach.

2.1 Identification Of Some Neuropathological Aging Markers In The Microcebus Murinus Brain. Bons N. (1), Jallageas V. (1), Privat N. (1), Mestre-Frances N. (1), Petter A. (2). (1). Neuromorphologie Fonctionnelle, Ecole Pratique des Hautes Etudes, Université Montpellier II, Place Bataillon, 34095 Montpellier, France. (2) 33 avenue Georges Clémenceau, 93160 Noisy-le-Grand, France. E-mail: ephemcb@crit.univ-montp2.fr

Keywords: Aging, Neuropathology, Tau proteins, beta amyloid, Neurotransmitters

Abstract: Behavioral changes have been observed in microcebe in captivity when older than the life expectancy in its natural habitat. These changes concern movement, social relations and in some cases, circadian rhythms. They are presumably the result of brain dysfunction. Microscopic and statistic analysis of microcebe brain sections from 1- to 13-year-old animals revealed three types of possible histo-pathological markers of aging. These observations were made in well-defined areas of brain sagittal sections with the same stereotaxic coordinates in all animals studied.

As previously demonstrated (1, 2) normal microtubule-associated Tau proteins in cortical neurons aggregate with age, due to hyperphosphorylation. The number of cortical pyramidal abnormally phosphorylated Tau protein-containing-neurons increased with age, and were five times more numerous in animals over 8 years old than in 1-7 years old animals.

In the brainstem, the number of neurons synthesizing various neurotransmitters fluctuated significantly with age. The catecholaminergic neuron count fell to 47% in the ventral tegmental area in 6-7- years old animals and 48% in the locus coeruleus in animals over 8-years old. The number of cholinergic neurons of the basal forebrain was very low in animals over 8 years old with neuronal alteration except for the diagonal band of Broca and the basal nucleus of Meynert where the loss was not as great.

In the cerebellum, the number of Purkinje cell decreased progressively with age to 25% of the maximum number in animals over 6 years old. In microcebes with numerous ß amyloid plaques, the Purkinje cell age-related loss increase was between 28% in 6-8 years old animals to 34% in 9-11 years-old microcebes.

The cortical parenchymal deposits of ß amyloid regularly observed in the ageing brain do not appear to be a marker of normal aging. We have previously described 4 stages in the evolution of these plaques (3). The earliest stage was often observed in the young animals studied. The more mature ß amyloid plaques depicted found only in animals over 5.5 years old did not increase with age. Large numbers of plaques were observed in 15% of the aged microcebes presumably constituting a pathological feature.

In conclusion, the numbers of cortical aggregated Tau-containing neurons, of the brainstem catecholaminergic elaborating neurons and of the Purkinje cells seem to be good indicators of the normal brain aging in Microcebus murinus. Consideration of ß amyloid plaques and/or cholinergic neuronal loss can be used to distinguish normal aging from age-related pathologies marked by aggravated neurological disorders.

References:
(1) Bons et al. (1995) Immunocytochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus. C.R.Acad. Sci. 318, 77-83.
(2) Delacourte et al. (1995) Biochemical characterization of Tau proteins during cerebral aging of the lemurian primate Microcebus murinus C.R. Acad. Sci. 318, 85-89.
(3) Mestre et al. (1996) Evolution of ß amyloid deposits in the cerebral cortex of the primate Microcebus murinus lemurian primate. Alzheimerís research 2, 19-28.

2.2 Nitric Oxide Synthase And Microglial Responses In An Animal Model Of Impaired Oxidative Metabolism. Noel Y. Calingasan, Chong H. Park, Leonard L. Calo and Gary E. Gibson. Cornell University Medical College at Burke Medical Research Institute, White Plains, NY 10605. E-mail: nycaling@mail.med.cornell.edu

Keywords: thiamine, amyloid precursor protein, free radicals, microglia, nitric oxide synthase

Abstract: Abnormal oxidative processes are a prominent feature of many neurodegenerative diseases including Alzheimer's disease (AD).Experimental thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative aberrations associated with thiamine-dependent enzyme deficits lead to selective neurodegeneration in brain. As in AD, TD is characterized by selective cell loss, reduced thiamine-dependent enzyme activities, cholinergic deficits and memory loss.

The exact mechanism responsible for the selective vulnerability in TD remains unclear. In rodent TD, breakdown of the blood-brain barrier (BBB) without alteration of interendothelial tight junction, is the earliest region-specific pathological change, preceding cell loss and accumulation of amyloid precursor protein and amyloid precursor-like protein 2 immunoreactivity in swollen, abnormal neurites and perikarya. The current studies tested the role of nitric oxide (NO) and microglia, two prominently proposed pathogenic mediators of neuronal loss in AD and other neurodegenerative diseases. Adult male rats or mice received a thiamine-deficient diet and daily injections of a thiamine antagonist. A region-specific increase in BBB permeability for IgG was accompanied by enhanced expression of endothelial nitric oxide synthase (NOS) immunoreactivity as well as NADPH diaphorase reactivity in blood vessels within vulnerable regions. The induction of NOS appears critical because immunoreactivity for nitrotyrosine, a specific nitration product of peroxynitrite, increased in axons within the thalamus, a vulnerable region.

Peroxynitrite is a potent oxidant that can be generated by reaction of superoxide with NO. In mouse TD, BBB breakdown was also accompanied by the occurrence of ferritin-positive activated microglia. In rats, large amounts of immunoreactive ferritin were detected in the walls of capillaries and larger blood vessels, as well as in microglia in the immediate vicinity of microvessels within the vulnerable brain regions. These observations support the hypothesis that a generalized impairment of oxidative metabolism increases NO and other compounds that generate reactive oxygen species in endothelial cells, and breaches the BBB in selective brain regions. Increases in the antioxidant protein ferritin in microvessel walls, as well as nitrotyrosine, and the presence of activated microglia suggest that oxidative damage is central to the pathogenesis of TD. We propose that factors released from endothelial cells and microglia such as NOS and other free radical generators act on metabolically compromised neurons and lead to neuronal death. Thus, the TD model may help to elucidate the relationship between oxidative deficits and BBB abnormalities, inflammatory response, ferritin elevation and selective cell loss, all of which occur in AD.