Luc Mercken

Amyloid Precursor Protein Family-induced Neuronal Death Is Mediated by Impairment of the Neuroprotective Calcium/Calmodulin Protein Kinase IV-dependent Signaling Pathway

The aberrant metabolism of β-amyloid precursor protein (APP) and the progressive deposition of its derived fragment β-amyloid peptide are early and constant pathological hallmarks of Alzheimers disease. Because APP is able to function as a cell surface receptor, we investigated here whether a disruption of the normal function of APP may contribute to the pathogenic mechanisms in Alzheimers disease. To this aim, we generated a specific chicken polyclonal antibody directed against the extracellular domain of APP, which is common with the β-amyloid precursor-like protein type 2. Exposure of cultured cortical neurons to this antibody (APP-Ab) induced cell death preceded by neurite degeneration, oxidative stress, and nuclear condensation. Interestingly, caspase-3-like protease was not activated in this neurotoxic action suggesting a different mode of cell death than classical apoptosis. Further analysis of the molecular mechanisms revealed a calpain- and calcineurin-dependent proteolysis of the neuroprotective calcium/calmodulin-dependent protein kinase IV and its nuclear target protein cAMP responsive element binding protein. These effects were abolished by the G protein inhibitor pertussis toxin, strongly suggesting that APP binding operates via a GTPase-dependent pathway to cause neuronal death.

Regulation of the amyloid precursor protein ectodomain shedding by the 5-HT4 receptor and Epac

The serotonin 5‐hydroxytryptamine (5‐HT4) receptor is of potential interest for the treatment of Alzheimers disease because it increases memory and learning. In this study, we investigated the effect of zinc metalloprotease inhibitors on the amyloid precursor protein (APP) processing induced by the serotonin 5‐HT4 receptor in vitro. We show that secretion of the non‐amyloidogenic form of APP, sAPPα induced by the 5‐HT4(e) receptor isoform was not due to a general boost of the constitutive secretory pathway but rather to its specific effect on α‐secretase activity. Although the h5‐HT4(e) receptor increased IP3 production, inhibition of PKC did not modify its effect on sAPPα secretion. In addition, we found that α secretase activity is regulated by the cAMP‐regulated guanine nucleotide exchange factor, Epac and the small GTPase Rac.

The caspase‐derived C‐terminal fragment of βAPP induces caspase‐independent toxicity and triggers selective increase of Aβ42 in mammalian cells

During its physiopathological maturation, the β‐amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimers disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Aβ40 and Aβ42, two highly aggregable βAPP‐derived fragments generated by combined cleavages by β‐ and γ‐secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids βAPP C‐terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31‐toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31‐stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine‐induced caspaseu20033‐like activity and pro‐caspase‐3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly‐(ADP‐ribose)‐polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Aβ42 but not Aβ40 production by HEK293 cells expressing wild‐type βAPP751. Altogether, our data demonstrate that C31 induces a caspase‐independent toxicity in TSM1 neurons and potentiates the pathogenic βAPP maturation pathway by increasing selectively Aβ42 species in wild type‐βAPP‐expressing human cells.

FE65 in Alzheimer’s Disease: Neuronal Distribution and Association with Neurofibrillary Tangles

FE65, a protein expressed in the nervous system, has the ability to bind the C-terminal domain of the amyloid precursor protein. This suggests a role for FE65 in the pathogenesis of Alzheimers disease (AD). The present study was conducted to find out if the distribution of FE65 immunoreactivity was affected during the course of AD, and to determine the degree of co-localization of FE65 with other proteins known to be involved in AD. Single immunoperoxidase-labeling experiments, conducted on six sporadic AD patients and six nondemented age-matched controls, showed that the proportion of volume occupied by FE65 immunoreactivity was not modified in the isocortex of AD patients. However, in hippocampal area CA4, increased FE65 immunoreactivity seemed to be associated with the severity of the disease. Double-immunofluorescent labeling did not show any clear co-localization of FE65 with the amyloid precursor protein. FE65 immunoreactivity was also absent from focal and diffuse deposits of the beta-amyloid peptide. Unexpectedly double labeling experiments showed a co-localization of FE65 and tau proteins in intracellular tangles. Ultrastructural observations confirmed that FE65 was associated with paired helical filaments.

Antibody-bound β-amyloid precursor protein stimulates the production of tumor necrosis factor-α and monocyte chemoattractant protein-1 by cortical neurons

Alzheimers disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar beta-amyloid (A beta), which is derived from the alternative processing of beta-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun N-terminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.

A risk for early-onset Alzheimer's disease associated with the APBB1 gene (FE65) intron 13 polymorphism.

Alzheimers disease (AD) is a genetically complex neurodegenerative disorder and the leading cause of dementia of the elderly. Recently, Hu et al. suggested that a trinucleotide deletion in intron 13 of the APBB1 gene was a factor protecting against late-onset AD. We report here the results of a case/control study aimed at replicating this association. Our study included 461 AD patients and 397 matched controls. We compared the allele and genotype frequencies of the polymorphism between the two groups but did not find any statistically significant difference (P=0.08 and P=0.09, respectively). By contrast, adjusting for age and sex, we found a slight risk associated with the deletion (odds ratio=1.47, 95% confidence interval=1.05-2.04). Stratification by age showed that the risk effect associated with the deletion concerned subjects aged less than 65 years.

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid β40

Frame‐shifted amyloid precursor protein (APP+1), which has a truncated out‐of‐frame C‐terminus, accumulates in the neuropathological hallmarks of patients with Alzheimers disease pathology. To study a possible involvement of APP+1 in the pathogenesis of Alzheimers disease, we expressed APP695 and APP+1 in the HEK293 cell‐line and studied whether the processing of APP695 was affected. APP+1 is a secretory protein, but high expression of APP695 and APP+1 results in the formation of intracellular aggregate‐like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP+1 is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post‐mortem brains of young Downs syndrome patients without any sign of neuropathology. Here we observed APP+1 immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4‐fold increase in levels of secreted amyloid β40 in cells co‐expressing APP695 and APP+1, although APP+1 itself does not contain the amyloid β sequence. Taken together, these data show that co‐expression of APP695 and APP+1 affects the processing of APP695 in a pro‐amyloidogenic way and this could gradually contribute to Alzheimers disease pathology, as has been implicated in Downs syndrome patients.

La protéine précurseur du peptide bêta-amyloïde : un système de transduction de la neuro-inflammation

La maladie d’Alzheimer (MA) est une pathologie neurodegenerative responsable d’un deficit cognitif d’evolution progressive. Elle se caracterise par la presence, dans l’hippocampe, l’amygdale temporale et la majorite du neocortex cerebral de 2 types de lesions : les plaques seniles et les degenerescences neurofibrillaires. Les plaques seniles sont caracterisees par un depot amyloide. Ce dernier est constitue principalement de la proteine beta-amyloide, peptides de 39 a 43 acides amines, produits du catabolisme d’un precurseur proteique membranaire, l’APP (amyloid protein precursor). Des modifications du metabolisme de l’APP, de la regulation du calcium intracellulaire, du metabolisme oxydatif et des systemes membranaires de transduction du signal ont ete impliquees dans la MA. Plusieurs etudes suggerent que les cytokines pro-inflammatoires, les chemokines et, de facon generale, la reponse inflammatoire jouent un role dans la pathogenie de la maladie. Nos etudes anterieures ont montre que l’utilisation d’un anticorps dirige contre le domaine extracellulaire de l’APP (APP-Ac) induit la mort cellulaire des neurones corticaux de souris. Dans cette etude, nous avons analyse la contribution eventuelle des cytokines et chemokines dans cette neurotoxicite de l’APP. Nos resultats montrent que APP-Ac induit l’expression des genes lies a l’inflammation au niveau transcriptionnel et traductionnel.