A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice.

The human antithrombin III-encoding gene (hAT-III) promoter (phAT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines. The human ApoA-II mRNA was detected at high levels, both in the liver and in the kidney of transgenic mice. The rat AT-III gene shows the same expression pattern. In contrast, as previously described, the same promoter permitted the expression of the SV40 large T antigen only in the liver of transgenic mice. In view of the extra-hepatic distribution of the ApoA-II mRNA, a preliminary characterization of the hAT-III proximal promoter (phAT-III), driving the expression of the transgene, was realized. Using DNase I footprinting analysis with liver nuclear extracts, four protected regions (I-IV) were identified in the first 175 bp of the 5' region of hAT-III. Electrophoretic mobility shift assays performed with liver and kidney nuclear extracts indicate that region III (nucleotides (nt) -67 to -90) interacts with the liver-enriched HNF4 nuclear factor. Furthermore, our data suggest that region I (nt -123 to -138) interacts with the liver-enriched HNF3 transcription factor family, both in liver and kidney. Taken together, these results demonstrate that phAT-III is a useful tool to create transgenic mice producing high plasma levels of a human apolipoprotein due to expression of the transgene in liver and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurofibrillary tangles and tau phosphorylation.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological lesion of Alzheimer's disease (AD). They are composed of a highly-phosphorylated form of the microtubule-associated protein tau. We are investigating the relationship between NFTs and microtubule stability and how tau phosphorylation and function is affected in transgenic models and by co-expression with beta-amyloid precursor protein and presenilins. In most NFT-bearing neurons, we observed a strong reduction in acetylated alpha-tubulin immunoreactivity (a marker of stable microtubules) and a reduction of the in situ hybridization signal for tubulin mRNA. In transfected cells, mutated tau forms (corresponding to tau mutations identified in familial forms of frontotemporal dementias linked to chromosome 17) were less efficient in their ability to sustain microtubule growth. These observations are consistent with the hypothesis that destabilization of the microtubule network is an important mechanism of cell dysfunction in Alzheimer's disease. The glycogen synthase kinase-3 beta (GSK-3 beta) generates many phosphorylated sites on tau. We performed a neuroanatomical study of GSK-3 beta distribution showing that developmental evolution of GSK-3 beta compartmentalization in neurons paralleled that of phosphorylated tau. Studies on transfected cells and on cultured neurons showed that GSK-3 beta activity controls tau phosphorylation and tau functional interaction with microtubules. Tau phosphorylation was not affected in neurons overexpressing beta-amyloid precursor protein. Transgenic mice expressing a human tau isoform and double transgenic animals for tau and mutated presenilin 1 have been generated; a somatodendritic accumulation of phosphorylated transgenic tau proteins, as observed in the pretangle stage in AD, has been observed but NFTs were not found, suggesting that additional factors might be necessary to induce their formation.

The metabolism of systemically-administered L-dihydroxyphenylalanine, by intact and dopamine-denervated striata, as revealed by brain microdialysis.

Idiopathic Parkinson's Disease arises from the progressive loss of dopamine (DA)-utilizing neurons of the nigrostriatum and responds to the replacement of DA with L-dihydroxyphenylalanine (L-DOPA). In awake rats, with unilateral lesions induced with 6-hydroxydopamine (6-OHDA) of the DA-utilizing nigrostriatal pathway, treatment with L-DOPA causes the rapid onset of brisk contralateral turning behaviour. In urethane-anesthetized rats with identical unilateral lesions of the nigrostriatum, dialysis of the striatum, performed before and after the systemic administration of L-DOPA (25 mg/kg i.p.), did not demonstrate any alteration in extracellular DA in the striatum which was DA-deprived compared to intact striata. After treatment with L-DOPA extracellular levels of the metabolites of DA. DOPAC and HVA increased several fold. These results suggest: (a) DA neurons surviving after extensive lesions with 6-OHDA can compensate for loss of DA in the striatum and maintain extracellular fluid (and presumably synaptic) concentrations of DA; (b) in striata with extensive depletion of DA L-DOPA undergoes rapid decarboxylation to DA, followed by catabolism to DOPAC and HVA; and (c) in urethane-anesthetized animals, DA formed from DOPA does not appear to enter a releasable pool.

Proteolytical processing of mutated human amyloid precursor protein in transgenic mice.

The evidence that betaA4 is central to the pathology of Alzheimer's disease (AD) came from the identification of several missense mutations in the amyloid precursor protein (APP) gene co-segregating with familial AD (FAD). In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of betaA4. Despite substantial amounts of betaA4 detected in the brain of the transgenic mice, neither signs of Alzheimer's disease-related pathology nor related behavioural deficits could be demonstrated.

Reduced antioxidant enzyme activity in brains of mice transgenic for human presenilin-1 with single or multiple mutations.

Alzheimer's disease-related mutations in the presenilin-1 gene (PS1) are leading to an elevated production of neurotoxic beta-amyloid 1-42 and may additionally enhance oxidative stress. Here, we provide in vivo evidence indicating that brains of transgenic mice expressing different human Alzheimer-linked PS1 mutations exhibit a reduced activity of two antioxidant enzymes. For this purpose, mice transgenic for human PS1 and for single and multiple PS1 mutations were generated. Mice with multiple PS1 mutations showed a significantly decreased activity of the antioxidant enzymes Cu/Zn superoxide dismutase and glutathione reductase already at an age of 3-4 months. As expected, this effect was less pronounced for the mice with a single PS1 mutation. By contrast, animals bearing normal human PS1 showed significantly elevated enzyme activities relative to non-transgenic littermate controls.

Increased tau phosphorylation but absence of formation of neurofibrillary tangles in mice double transgenic for human tau and Alzheimer mutant (M146L) presenilin-1.

Neurofibrillary tangles, composed of tau proteins, are a key lesion observed in sporadic forms of Alzheimer's disease and in familial forms associated with mutations of presenilin-1 (PS1). We have generated a double transgenic mouse line expressing a human tau isoform and a mutated form of PS1 (M146L) in neurons. Increased expression of the PS1 holoprotein was observed in the tau/PS1 transgenic mice and the proteolytic fragments of PS1 did not appear to be modified. A somatodendritic accumulation of the transgenic tau and an increase in tau phosphorylation were observed in both tau- and tau/PS1 transgenic mice. Neurofibrillary tangles were not observed in animals analyzed up to 17 months. Immunoprecipitation of tau from brain homogenates demonstrated its binding with active glycogen synthase kinase-3beta in control, tau- and tau/PS1 transgenic lines. These results suggest that overexpression of this Alzheimer mutant PS1 in vivo is not by itself sufficient to induce the formation of neurofibrillary tangles, even in neurons co-expressing and accumulating a human tau isoform.

D1 dopamine agonist effects assessed in vivo with [14C]-2-deoxyglucose autoradiography.

In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra, the 2-deoxyglucose (2-DG) autoradiographic method of measuring regional cerebral glucose utilization (RCGU) was used to assess the effects of three systemically administered dopamine agonists: bromocriptine, pergolide and (+)-4-propyl-9-hydroxynaphoxazine (PHNO). Pergolide increased RCGU in the substantia nigra pars reticulata (SNr) ipsilateral to the lesion in a dose-dependent manner (0.04 mg/kg, up 52%; 0.4 mg/kg, up 111%), resulting in asymmetric glucose utilization on the dopamine-denervated and intact sides of the brain. Pretreatment with a selective D1 antagonist (SCH 23390, 0.5 mg/kg) blocked completely the RCGU increase elicited by pergolide (0.4 mg/kg) whereas pretreatment with a selective D2 antagonist (eticlopride, 1.0 mg/kg) only mildly attenuated this increase. The effect of drug treatments on RCGU in the entopeduncular nucleus (EP) paralleled that in the SNr. These results demonstrate that the RCGU increase in the EP and SNr after pergolide administration is dependent primarily on D1 receptor stimulation. Administration of bromocriptine and PHNO minimally altered RCGU in the ipsilateral EP and SNr and did not result in significant left/right RCGU asymmetry. Considered in the context of prior studies of selective D1 and D2 agonists, the results suggest that, in this model, the magnitude of the RCGU increase in the EP and SNr elicited by a dopamine agonist, above the modest effects produced by selective D2 stimulation, represents a measure of D1 agonist effect in vivo. The results support a nonselective D1/D2 stimulatory effect of pergolide (0.04-0.4 mg/kg) and a selective D2 action of both bromocriptine and PHNO.

Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer's disease mutants of APP and presenilin-1.

To study the role of Abeta amyloid deposits in the generation of cytoskeletal lesions, we have generated a transgenic mouse line coexpressing in the same neurons a wild-type human tau isoform (0N3R), a mutant form of APP (751SL) and a mutant form of PS1 (M146L). These mice developed early cerebral extracellular deposits of Abeta, starting at 2.5 months. A somatodendritic neuronal accumulation of transgenic tau protein was observed in tau only and in tau/PS1/APP transgenic mice, including in neurons adjacent to Abeta deposits. The phosphorylation status of this somatodendritic tau was similar in the two transgenic lines. The Abeta deposits were surrounded by a neuritic reaction composed of axonal dystrophic processes, immunoreactive for many phosphotau epitopes and for the human tau transgenic protein. Ultrastructural observation showed in these dystrophic neurites a disorganisation of the microtubule and the neurofilament network but animals that were observed up to 18 months of age did not develop neurofibrillary tangles. These results indicate that overexpression of mutant PS1, mutant APP and of wild-type human tau were not sufficient per se to drive the formation of neurofibrillary tangles in a transgenic model. The Abeta deposits, however, were associated to marked changes in cytoskeletal organisation and in tau phosphorylation in adjacent dystrophic neurites.

Alzheimer's disease-like alterations in peripheral cells from presenilin-1 transgenic mice.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Expression of PS1 mutations in cell culture systems and in primary neurons from transgenic mice increases their vulnerability to cell death. Interestingly, enhanced vulnerability to cell death has also been demonstrated for peripheral lymphocytes from AD patients. We now report that lymphocytes from PS1 mutant transgenic mice show a similar hypersensitivity to cell death as do peripheral cells from AD patients and several cell culture systems expressing PS1 mutations. The cell death-enhancing action of mutant PS1 was associated with increased production of reactive oxygen species and altered calcium regulation, but not with changes of mitochondrial cytochrome c. Our study further emphasizes the pathogenic role of mutant PS1 and may provide the fundamental basis for new efforts to close the gap between studies using neuronal cell lines transfected with mutant PS1, neurons from transgenic animals, and peripheral cells from AD patients.

Interleukin-1 beta-specific partial agonists defined by site-directed mutagenesis studies.

Monocyte-derived interleukin 1 (IL-1) mediates a wide range of biological effects including destruction of the cartilage matrix in articular diseases such as rheumatoid and osteoarthritis. To elucidate further the relationships between protein structure and biological activities, we have analyzed the sequence of several IL-1 polypeptides using the algorithm of Parker, the hydrophobic cluster analysis method and published structural data. This led us to identify several residues that seemed to be strictly topologically conserved, with respect to identifiable secondary structures features, although this was not readily apparent from sequence alignments. We performed site-directed mutagenesis on some of these conserved residues, as well as on those predicted to occur in external loops of the polypeptide. Human IL-1 beta mutant polypeptides were expressed in Escherichia coli in soluble form and purified to homogeneity by anion-exchange and gel-filtration chromatography. Their biological effects (binding to EL4-6.1 murine thymocytes, Raji human B cells and rabbit chondrocytes cells, lymphocyte activation, neutral protease induction, proteoglycan degradation and synthesis) have been determined. Among the 20 IL-1 beta mutant polypeptides we present here, four showed a markedly reduced activity in cartilage matrix assays without any significant change in their binding to the cartilage matrix cells (chondrocytes). Furthermore, some of these mutants were specific partial agonists of the effects of IL-1 on connective tissue since they have a low affinity for thymocytes.