Electrophysiological studies on the hippocampus and prefrontal cortex assessing the effects of amyloidosis in amyloid precursor protein 23 transgenic mice.

In vitro and in vivo electrophysiological studies were done to investigate the neuronal function of the hippocampus and prefrontal cortex in the amyloid precursor protein (APP) 23 transgenic mouse model for amyloidosis developed by Sturchler-Pierrat et al. [Proc Natl Acad Sci USA 94 (1997) 13287]. Brain slices were taken from 3, 6, 9, 12, 18 and 24 month old wildtype and hemizygous type APP23 mice. Extracellular field potentials were recorded from the CA1 region of the hippocampus while stimulating the Schaffer collaterals. In addition, extracellular field potentials were elicited from areas within layer V/VI of the prefrontal cortex by stimulating the same layer V/VI. Basic synaptic function in the hippocampus was reduced in hemizygous APP23 mice compared with their wildtype littermates at 12 and 18 months of age, whereas, it was unaltered within the prefrontal cortex. Long-term potentiation in the hippocampus and the prefrontal cortex of hemizygous APP23 mice was similar compared with their wildtype littermates. In vivo electrophysiological experiments were done in 3, 9, 18 and 24 month old wildtype and hemizygous APP23 mice. No differences were observed in the number of single spontaneously active units recorded within the prefrontal cortex of hemizygous APP23 mice compared with their wildtype littermates. Field potentials elicited during stimulation of cortico-cortical pathways to assess synaptic transmission and short-term synaptic plasticity were also unchanged in hemizygous APP23 mice. Furthermore, presumable antidromic field potentials recorded in the prefrontal cortex during stimulation of the striatum were similar between the hemizygous APP23 and wildtype mice at each age. The present study shows that amyloidosis impairs basic synaptic function but not long-term potentiation in the hippocampus, however, does not alter any of the neurophysiological functions measured within the prefrontal cortex. These findings suggest that amyloidosis may be involved in altering some neurophysiological functions within only certain brain structures. Although APP23 mice have impaired cognitive performance, long-term plasticity, a cellular model for memory, is not affected, raising the question on the relationship between these processes.

Progressive age-related impairment of cognitive behavior in APP23 transgenic mice.

Transgenic APP23 mice expressing human APP(751) with the K670N/M671L mutation, were compared at ages 3, 18 or 25 months to non-transgenic littermates in passive avoidance and in a small and large Morris maze. The task in the smaller pool habituated their flight response to the platform. Impairments in passive avoidance and small pool performance in APP23 mice were clearly age-related. In the larger Morris maze APP23 mice at all ages were impaired in latency and distance swum before finding the platform. Identical performance of 18-month APP23 and controls in a visible platform condition indicates that the Morris maze performance deficit was not due to sensory, motor or motivational alterations. At age 3 months both groups initially unexpectedly avoided the visible platform, suggesting that in young mice neophobia may contribute significantly to performance in cognitive tests. In conclusion, APP23 mice exhibit both early behavioral impairment in the large Morris maze as well as impairments in passive avoidance and small pool performance that are marked only in old age.

Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy.

A high risk factor for spontaneous and often fatal lobar hemorrhage is cerebral amyloid angiopathy (CAA). We now report that CAA in an amyloid precursor protein transgenic mouse model (APP23 mice) leads to a loss of vascular smooth muscle cells, aneurysmal vasodilatation, and in rare cases, vessel obliteration and severe vasculitis. This weakening of the vessel wall is followed by rupture and bleedings that range from multiple, recurrent microhemorrhages to large hematomas. Our results demonstrate that, in APP transgenic mice, the extracellular deposition of neuron-derived beta-amyloid in the vessel wall is the cause of vessel wall disruption, which eventually leads to parenchymal hemorrhage. This first mouse model of CAA-associated hemorrhagic stroke will now allow development of diagnostic and therapeutic strategies.

Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice.

A microglial response is part of the inflammatory processes in Alzheimer's disease (AD). We have used APP23 transgenic mice overexpressing human amyloid precursor protein with the Swedish mutation to characterize this microglia response to amyloid deposits in aged mice. Analyses with MAC-1 and F4/80 antibodies as well as in vivo labeling with bromodeoxyuridine demonstrate that microglia in the plaque vicinity are in an activated state and that proliferation contributes to their accumulation at the plaque periphery. The amyloid-induced microglia activation may be mediated by scavenger receptor A, which is generally elevated, whereas the increased immunostaining of the receptor for advanced glycation end products is more restricted. Although components of the phagocytic machinery such as macrosialin and Fc receptors are increased in activated microglia, efficient clearance of amyloid is missing seemingly because of the lack of amyloid-bound autoantibodies. Similarly, although up-regulation of major histocompatibility complex class II (IA) points toward an intact antigen-presenting function of microglia, lack of T and B lymphocytes does not indicate a cell-mediated immune response in the brains of APP23 mice. The similar characteristics of microglia in the APP23 mice and in AD render the mouse model suitable to study the role of inflammatory processes during AD pathogenesis.

Mouse models of alpha-synucleinopathy and Lewy pathology.

The discovery of two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein alpha-synuclein (alphaSN) that are genetically linked to rare familial forms of Parkinson's disease and its accumulation in Lewy bodies and Lewy neurites has triggered several attempts to generate transgenic mice overexpressing human alphaSN. Analogous to a successful strategy for the production of transgenic animal models for Alzheimer's disease we generated mice expressing wildtype and the A53T mutant of human alphaSN in the nervous system under control of mouse Thy1 regulatory sequences. These animals develop neuronal alpha-synucleinopathy, striking features of Lewy pathology, neuronal degeneration and motor defects. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions, suggesting that alphaSN may interfere with a universal mechanism of synapse maintenance. Thy1-transgene expression of wildtype human alphaSN resulted in comparable pathological changes thus supporting a central role for mutant and wildtype alphaSN in familial and idiopathic forms of diseases with neuronal alpha-synucleinopathy and Lewy pathology. The mouse models provide means to address fundamental aspects of alpha-synucleinopathy and to test therapeutic strategies.

Neuropathology in mice expressing human alpha-synuclein.

The presynaptic protein alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. alpha-Synuclein accumulates in Lewy bodies and Lewy neurites, and two missense mutations (A53T and A30P) in the alpha-synuclein gene are genetically linked to rare familial forms of Parkinson's disease. Under control of mouse Thy1 regulatory sequences, expression of A53T mutant human alpha-synuclein in the nervous system of transgenic mice generated animals with neuronal alpha-synucleinopathy, features strikingly similar to those observed in human brains with Lewy pathology, neuronal degeneration, and motor defects, despite a lack of transgene expression in dopaminergic neurons of the substantia nigra pars compacta. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions in several muscles examined, suggesting that alpha-synuclein interfered with a universal mechanism of synapse maintenance. Thy1 transgene expression of wild-type human alpha-synuclein resulted in similar pathological changes, thus supporting a central role for mutant and wild-type alpha-synuclein in familial and idiotypic forms of diseases with neuronal alpha-synucleinopathy and Lewy pathology. These mouse models provide a means to address fundamental aspects of alpha-synucleinopathy and test therapeutic strategies.

Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein.

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some of these mutations lead to an overproduction of tau isoforms with four microtubule-binding repeats. Here we have expressed the longest four-repeat human brain tau isoform in transgenic mice under the control of the murine Thy1 promoter. Transgenic mice aged 3 weeks to 25 months overexpressed human tau protein in nerve cells of brain and spinal cord. Numerous abnormal, tau-immunoreactive nerve cell bodies and dendrites were seen. In addition, large numbers of pathologically enlarged axons containing neurofilament- and tau-immunoreactive spheroids were present, especially in spinal cord. Signs of Wallerian degeneration and neurogenic muscle atrophy were observed. When motor function was tested, transgenic mice showed signs of muscle weakness. Taken together, these findings demonstrate that overexpression of human four-repeat tau leads to a central and peripheral axonopathy that results in nerve cell dysfunction and amyotrophy.

Transgenic approaches to model Alzheimer's disease.

Two transgenic mouse lines were generated which express human APP751 containing familial Alzheimer's disease (AD) mutations in brain neurons. These mice develop pathological features reminiscent of AD. The degree of pathology depends on both expression levels and specific mutations. In mice with more advanced pathology (APP 23), typical plaques appear at six months which increase with age and are Congo Red positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. A quantitative analysis of degenerative changes by state-of-the-art unbiased stereological methods revealed a significant reduction in neuronal cell bodies of the CA1 field of the hippocampus when compared to controls. This reduction is directly related to plaque load. When subjected to analysis in the Morris water maze, 18 month old APP 23 mice show a significant increase in platform finding latency throughout the entire trial when compared to non-transgenic littermates.

Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid.

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer's disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid beta (Abeta) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer's disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Abeta in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Abeta as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Abeta is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (A beta) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes A beta deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A beta in the pathogenesis of the disease.

Cloning, distribution and functional expression of the human mGlu6 metabotropic glutamate receptor.

The cDNA encoding the human metabotropic glutamate receptor type 6 (hmGlu6) was isolated from a human retinal cDNA library. The deduced primary sequence (877 amino acids) of the hmGlu6 receptor was 93.5% identical to its rat counterpart and shared 69.8% sequence identity with the related hmGlu4 receptor clone (912 amino acids), isolated in parallel from a human brain cDNA library. In situ hybridization revealed that the hmGlu6 mRNA is highly expressed in cells located in the inner nuclear layer of the human retina, presumably bipolar neurons. Neither PCR analysis nor in situ hybridization could detect hmGlu6 mRNA in human brain. When stably expressed in Chinese hamster ovary cells (CHO-K1) the hmGlu6 receptor inhibited adenylate cyclase through a pertussis toxin-sensitive G-protein, and reduced forskolin-elevated cyclic adenosine monophosphate (cAMP) levels in response to agonists. The rank order of agonist potency was L(+)-2-amino-4-phosphonobutyric acid (L-AP4) > L-serine-O-phosphate > L-glutamate > quisqualate = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD). (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I) was a partial agonist at the hmGlu6 receptor, with a potency approaching that of L-serine-O-phosphate.

Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram.

1. We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF-alpha) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2. After intravenous administration of LPS, a similar time-dependent induction of both TNF-alpha mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3. In situ hybridization experiments with a specific antisense TNF-alpha riboprobe suggested that the cells responsible for TNF-alpha production in the brain were microglia. 4. Intraperitoneal administration of methylprednisolone inhibited the induction of TNF-alpha protein in a dose-dependent manner. A maximal inhibition of TNF-alpha protein production by 42.9+/-10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg(-1) methylprednisolone. 5. Intraperitoneal administration of rolipram also inhibited the induction of TNF-alpha protein in a dose-dependent manner. The maximal inhibition of TNF-alpha protein production was 96.1+/-12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg(-1) rolipram. 6. In situ hybridization experiments showed that the level of TNF-alpha mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2 x 15 mg kg(-1)) and of rolipram (3 x 3 mg kg(-1)). 7. We suggest that peripheral administration of LPS induces a time-dependent expression of TNF-alpha in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS-induced expression of TNF-alpha in these cells via a decrease of TNF-alpha mRNA stability and/or TNF-alpha gene transcription.

Biphenyl-derivatives of 2-amino-7-phosphono-heptanoic acid, a novel class of potent competitive N-methyl-D-aspartate receptor antagonists--II. Pharmacological characterization in vivo.

A selection of biphenyl-analogues of 2-amino-7-phosphonoheptanoic acid (AP7), N-methyl-D-aspartate (NMDA) receptor antagonists with high affinity in vivo efficacy. The lead compound SDZ EAB 515 was found to inhibit L-phenylalanine uptake by the large neutral amino acid carrier in vitro and in vivo; active transport may thus confer a good bioavailability to this class of compounds. CNS effects were demonstrated by significant changes in 2-deoxyglucose-uptake in various brain regions at doses from 1 to 10 mg/kg i.p. With the most active agent, SDZ 220-581, full protection against maximal electroshock seizures (MES) was obtained at oral doses of 10 mg/kg in rats and in mice. The compound had a fast onset (< or = 1 hr) and a long duration (> or = 24 hr) of action. Motor-debilitating effects (impairment of rotarod performance) occurred at doses about 10 times higher than those required for protection against MES. Neuroprotective activity was demonstrated by the ability of the compounds to reduce the extent of quinolinic acid-induced striatal lesions in rats, in the dose range of 3-15 mg/kg (i.p.) or 10-50 mg/kg (p.o.). In the middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats, the test compounds reduced the infarct size by 40-50% when given i.v. before or by 20-30% when given i.v. 1 hr after MCAO. SDZ 220-581 provided 20-30% protection at > or = 2 x 10 mg/kg p.o. This compound also showed analgesic activity at low oral doses in a model of neuropathic pain, although higher doses were required in model of mechanical inflammatory hyperalgesia. Unexpectedly, SDZ 220-581 at low s.c. doses counteracted the antiparkinsonian effects of L-DOPA in MPTP-treated marmosets. (Sub)chronic administration of SDZ 220-581 did not reduce its ability to protect against quinolinic acid neurotoxicity, and no upregulation of NMDA receptors was detected using a [3H]CGP-39653 binding assay. In conclusion, from a series of biphenyl-AP7-derivatives, SDZ 220-581 is clearly the most active compound in vivo. Its pharmacological profile with a good, long-lasting oral activity might open up novel therapeutic applications for competitive NMDA receptor antagonists.

Expression of APP in transgenic mice: a comparison of neuron-specific promoters.

The beta-amyloid precursor protein (APP) carries mutations in codons 717 or 670/671, which cosegregate with familial forms of Alzheimer's disease (AD). As an initial step to study the related pathogenetic mechanisms in vivo we have generated transgenic mice expressing APP with these mutations. Several neuron-specific promoters were used to drive expression of human APP cDNAs. Only the Thy-1 promoter yielded transgene expression levels comparable to or above the endogenous mouse levels. Deletion of a 121 bp sequence from the 3' untranslated region of APP appeared to increase mRNA levels. Transgene mRNA was found throughout the brain with highest levels in hippocampus and cerebral cortex. Accordingly, human APP was detected in these regions by Western blotting. Protein levels paralleled mRNA levels reaching or exceeding the amount of endogenous APP. Variable reactivity of human APP in cell bodies was shown by immunocytochemistry. Although our initial histological examinations did not reveal any alterations characteristic of AD, further studied will be required.

Inhibitors of type IV phosphodiesterases reduce the toxicity of MPTP in substantia nigra neurons in vivo.

The neuropathology of Parkinson's disease is characterized by the degeneration of dopaminergic neurons in the substantia nigra. We have recently shown that the activation of protein kinase A improves the survival of dopaminergic neurons in culture and, furthermore, protects them from the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) in vitro. We have now analysed the potential of phosphodiesterase inhibitors to increase cAMP levels in dopaminergic neurons, to improve their survival in culture and to protect them from the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo. Increasing intracellular cAMP with phosphodiesterase type IV-specific inhibitors enhanced the survival of dopaminergic neurons in culture. Inhibitors of other phosphodiesterase types were not active. In vivo, phosphodiesterase type IV inhibitors reduced the MPTP-induced dopamine depletion in the striatum of C57BL/6 mice. Furthermore, the loss of tyrosine hydroxylase-immunopositive neurons in the substantia nigra of these animals was diminished. After Nissl staining, a similar reduction of the MPTP-induced loss of neurons was observed in the substantia nigra. The protective effect of protein kinase A activation did not appear to be due to the blocking of MPP+ uptake into dopaminergic neurons. This was not decreased after treatment with forskolin or 8-(4-chlorophenylthio)-cAMP. Thus, protein kinase A regulates the survival and differentiation of dopaminergic substantia nigra neurons in vivo, implicating a therapeutic potential for substances which regulate cAMP turnover in these neurons.

Stereoisomerism and muscarinic receptor agonists: synthesis and effects of the stereoisomers of 3-[5-(3-amino-1,2,4-oxadiazol)yl]-1- azabicyclo[2.2.1]heptane.

The preparation and the biological activities of the four stereoisomers of 3-[5-(3-amino-1,2,4-oxadiazol)yl]-1-azabicyclo[2.2.1]heptane are described. The most potent stereoisomer, 3a, has the 3R,4R configuration, and in vitro activities in (pD2(% efficacy): ileum 8.8 (87%), hippocampus 9.8 (116%) and ganglion 10.2 (36%)). 3b (3S,4S) was weaker (ileum 8.1 (121%), hippocampus 8.5 (107%), ganglion 9.0 (63%)). The other two stereoisomers, 4a (3S,4R; ileum 7.1 (108%), hippocampus 8.2 (116%), ganglion 7.3 (31%)) and 4b (3R,4S; ileum 7.0 (100%), hippocampus 7.0 (120%), ganglion 7.2 (67%)) are of comparable activity, with an analogous profile to that of the more potent stereoisomers. Thus, compounds 3a and 4a, possessing the 4R stereochemistry, showed selectivity for the hippocampus over the ileum. Compound 3a was, however, more potent in the ganglion than in the hippocampus. All four stereoisomers were full agonists in the hippocampus, indicating M1 activity; however, they were partial agonists in the depolarisation of the rat superior cervical ganglion, another M1-mediated response. This may be due to M2-mediated hyperpolarization. With 3a (0.01 mg/kg i.p.), expression of c-fos mRNA was observed in the hypothalamus and in brain areas involved in sensory processing; these effects were totally blocked by pretreatment with 2 mg/kg scopolamine. In particular, activation of the superior colliculus is consistent with potent M2 activity.

Intracerebroventricular application of competitive and non-competitive NMDA antagonists induce similar effects upon rat hippocampal electroencephalogram and local cerebral glucose utilization.

In this study we have used electrophysiological and metabolic markers to investigate the effects of competitive and non-competitive NMDA antagonists in rats after central or peripheral administration. The non-competitive antagonist, MK-801, induced dose-dependent suppression of rat hippocampal EEG energy both after intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) application. Similar effects were observed after i.p. and i.c.v. application of the competitive antagonist, DL-CPP-ene. Whereas the MK-801 was more potent after i.p. application, DL-CPP-ene was more potent after i.c.v. administration. Intracerebroventricular administration of MK-801 and DL-CPP-ene resulted in similar changes in the pattern of local cerebral glucose utilization in the olfactory tubercle and regions of the limbic system such as the anteroventral thalamus, hippocampus and entorhinal cortex. Intravenous (i.v.) administration of MK-801 induced increases in glucose metabolism similar to those observed after i.c.v. application. In contrast, i.v. administration of DL-CPP-ene induced only small decreases of glucose utilization in several regions of the central sensory system. Thus the blockade of glutamatergic (NMDA) transmission results in decreased hippocampal EEG activity which is paralleled by increased metabolic activity in this area. We conclude from EEG recordings and [14C]2-deoxyglucose uptake experiments that both non-competitive and competitive NMDA antagonists produce the same pattern of alterations after i.c.v. administration. Apparent differences in efficacy after peripheral administration may be largely due to differences in bioavailability.

Muscarinic M2-selective ligands also recognize M4 receptors in the rat brain: evidence from combined in situ hybridization and receptor autoradiography.

We have used autoradiographic techniques to examine the characteristics and distribution of the binding of reported selective M2 muscarinic ligands and compared them with the distribution of cells expressing mRNAs for the different subtypes of muscarinic receptors. Our results suggest that the M2 ligands used in the present study ([3H]OXO-M, ([3H]OXO-M,[3H]AF-DX384,AF-DX116, methoctramine) also recognize M4 receptors present in regions such as the striatum and olfactory tubercle. This is supported by 1) relative abundances of the different transcripts, with m2 mRNA being very scarce and m4 mRNA very abundant in these regions; 2) comparison of the pharmacological characteristics of M2-ligand binding sites in brain areas selected by their exclusive expression of M2 receptors versus areas enriched in M4 receptors. An important conclusion of these studies is that none of the muscarinic radioligands available at the present time appears to label specifically a single muscarinic receptor subtype population. Areas are suggested where autoradiographic techniques can be helpful in elucidating the subtype selectivity of existing and new ligands.