Véronique Blanchard

Massive CA1/2 Neuronal Loss with Intraneuronal and N-Terminal Truncated Aβ42 Accumulation in a Novel Alzheimer Transgenic Model

Alzheimers disease (AD) is characterized by a substantial degeneration of pyramidal neurons and the appearance of neuritic plaques and neurofibrillary tangles. Here we present a novel transgenic mouse model, APP(SL)PS1KI that closely mimics the development of AD-related neuropathological features including a significant hippocampal neuronal loss. This transgenic mouse model carries M233T/L235P knocked-in mutations in presenilin-1 and overexpresses mutated human beta-amyloid (Abeta) precursor protein. Abeta(x-42) is the major form of Abeta species present in this model with progressive development of a complex pattern of N-truncated variants and dimers, similar to those observed in AD brain. At 10 months of age, an extensive neuronal loss (>50%) is present in the CA1/2 hippocampal pyramidal cell layer that correlates with strong accumulation of intraneuronal Abeta and thioflavine-S-positive intracellular material but not with extracellular Abeta deposits. A strong reactive astrogliosis develops together with the neuronal loss. This loss is already detectable at 6 months of age and is PS1KI gene dosage-dependent. Thus, APP(SL)PS1KI mice further confirm the critical role of intraneuronal Abeta(42) in neuronal loss and provide an excellent tool to investigate therapeutic strategies designed to prevent AD neurodegeneration.

Intraneuronal Aβ accumulation precedes plaque formation in β-amyloid precursor protein and presenilin-1 double-transgenic mice

beta-Amyloid peptides are key molecules that are involved in the pathology of Alzheimers disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversial debates. In the present report, we studied the neuropathological events in a transgenic mouse model expressing human mutant beta-amyloid precursor protein and human mutant presenilin-1 in neurons. Western blot and immunohistochemical analysis revealed that intracellular Abeta staining preceded plaque deposition, which started in the hippocampal formation. At later stages, many neuritic Abeta positive plaques were found in all cortical, hippocampal and many other brain areas. Interestingly, intraneuronal Abeta staining was no longer detected in the brain of aged double-transgenic mice, which correlates with the typical neuropathology in the brain of chronic AD patients.

Time sequence of maturation of dystrophic neurites associated with Aβ deposits in APP/PS1 transgenic mice

Several novel transgenic mouse models expressing different mutant APPs in combination with mutant PS1 have been developed. These models have been analyzed to investigate the formation and progressive alterations of dystrophic neurites (DNs) in relation to Abeta deposits. In the most aggressive model, Abeta deposits appear as early as 2.5 months of age. Maturation of DNs was qualitatively quite similar among models and in some respect reminiscent of human AD pathology. From the onset of deposition, most if not all Abeta deposits were decorated with a high number of APP-, ubiquitin-, and MnSOD-immunoreactive DNs. Phosphorylated Tau DNs, however, appeared at a much slower rate and were more restricted. Mitochondrial dysfunction markers were observed in DNs: the frequency and the density per deposit of DNs accumulating cytochrome c, cytochrome oxidase 1, and Bax progressively increased with age. Later, the burden of reactive DNs was reduced around large compact/mature deposits. In addition, the previously described phenomenon of early intraneuronal Abeta accumulation in our models was associated with altered expression of APP protein as well as oxidative and mitochondrial stress markers occasionally in individual neurons. The present study demonstrates that oxidative and mitochondrial stress factors are present at several phases of Abeta pathology progression, confirming the neuronal dysfunction in APP transgenic mice.

Subcellular topography of neuronal Aβ peptide in APPxPS1 transgenic mice

In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.

Intraneuronal APP/Aβ Trafficking and Plaque Formation in β-Amyloid Precursor Protein and Presenilin-1 Transgenic Mice

Neuropil deposition of β‐amyloid peptides Aβ40 and Aβ42 is believed to be the key event in the neurodegenerative processes of Alzheimers disease (AD). Since Aβ seems to carry a transport signal that is required for axonal sorting of its precursor β‐amyloid precursor protein (APP), we studied the intraneuronal staining profile of Aβ peptides in a transgenic mouse model expressing human mutant APP751 (KM670/671NL and V717I) and human mutant presenilin‐1 (PS‐1 M146L) in neurons. Using surface plasmon resonance we analyzed the Aβ antibodies and defined their binding profile to APP, Aβ40 and Aβ42. Immunohistochemical staining revealed that intraneuronal Aβ40 and Aβ42 staining preceded plaque deposition, which started at 3 months of age. Aβ was observed in the somatodendritic and axonal compartments of many neurons. Interestingly, the striatum, which lacks transgenic APP expression harbored many plaques at 10 months of age. This is most likely due to an APP/Aβ transport problem and may be a model region to study APP/Aβ trafficking as an early pathological event.

The Antioxidant Ebselen Prevents Neurotoxicity and Clinical Symptoms in a Primate Model of Parkinson's Disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), via its major metabolite 1-methyl-4-phenylpyridinium (MPP(+)), produces in primates including humans clinical, biochemical, and neuropathological changes similar to those which occur in idiopathic Parkinsons disease. Ebselen is an antioxidant drug with glutathione peroxidase-like activity and a proven neuroprotective action in stroke patients. Here we show that Ebselen, when administered before, during, and after MPTP injections, prevents both neuronal loss and clinical symptoms in a primate MPTP model of Parkinsons disease. Ebselen also prevents peroxide radical overproduction induced by serum withdrawal in cultured PC12 cells and hydroxyl radical generation induced by the mitochondrial toxin, MPP(+), in vivo in rat brain. Moreover, Ebselen inhibits MPP(+)-induced toxicity in PC12 cells, without interacting with the dopamine uptake system. Our results demonstrate that compounds which prevent mitochondrial dysfunction and free radical production may be useful as preventive treatment of Parkinsons disease.

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.

Reelin in plaques of β-amyloid precursor protein and presenilin-1 double-transgenic mice

There is circumstantial evidence that the reelin signaling pathway may contribute to neurodegeneration in the adult brain and could be linked to Alzheimers disease (AD). In the present immunohistochemical report we studied the reelin expression profile in double-transgenic mice that express both human mutant beta-amyloid precursor protein (APP) and human mutant presenilin-1. We were able to demonstrate that reelin immunostaining was found together with human APP in the neuritic component of many AD-typical plaques in both hippocampus and neocortex. This observation gives the first evidence for the association of reelin with amyloid deposits.

An in vitro and in vivo study of early deficits in associative learning in transgenic mice that over‐express a mutant form of human APP associated with Alzheimer's disease

Transgenic mice over‐expressing a mutated form of the human amyloid precursor protein (APP, 695 isoform) bearing a mutation associated with Alzheimers disease (V642I, so‐called London mutation, hereafter APPLd2) and wild‐type controls were studied at age periods (3 and 10 months) prior to the overt development of neuritic amyloid plaques. Both 3‐ and 10‐month‐old APPLd2 mice had reflex eyelid responses like those of controls, but only younger mice were able to acquire a classical conditioning of eyelid responses in a trace paradigm. In vitro studies on hippocampal slices showed that 10‐month‐old APPLd2 mice also presented deficits in paired‐pulse facilitation and long‐term potentiation, but presented a normal synaptic activation of CA1 pyramidal cells by the stimulation of Schaffer collaterals. It is proposed that definite functional changes may appear well in advance of noticeable structural alterations in this animal model of Alzheimers disease, and that specific learning tasks could have a relevant diagnostic value.

Neuroprotective effect of riluzole in a primate model of Parkinson's disease: Behavioral and histological evidence

Our study aimed to determine whether riluzole, which has shown efficacy as a disease‐modifying agent in amyotrophic lateral sclerosis (ALS), is neuroprotective in a marmoset model of Parkinsons disease (PD). Reduction of energy demand by riluzole could be a rational neuroprotective strategy with good tolerability. The efficacy of riluzole was evaluated in marmosets by testing its ability to reduce MPTP‐induced behavioral deficits and loss of dopaminergic nigral neurons. Marmosets were divided into two groups of four animals each: animals in Group 1 were injected twice with MPTP (2 mg/kg subcutaneous) and treated with riluzole (10 mg/kg per os b.i.d.), animals in Group 2 (controls) were injected with MPTP and with the vehicle of riluzole. A third group of marmosets which did not receive MPTP or riluzole drug was introduced for neurohistopathological studies (normal animals). Marmosets treated with riluzole preserved a better motor function and neurological performance through the 26 days of assessment when compared with the controls. Histologically, there was sparing of TH‐ and Nissl‐stained nigral neurons and of TH‐stained terminals in the striatum and the putamen in the group treated with riluzole compared to the controls. We conclude that riluzole protects dopaminergic neurons and reduces behavioral deficits in a marmoset model of PD.