Moustapha Cissé

Reversing EphB2 depletion rescues cognitive functions in Alzheimer model

Amyloid-β oligomers may cause cognitive deficits in Alzheimer’s disease by impairing neuronal NMDA-type glutamate receptors, whose function is regulated by the receptor tyrosine kinase EphB2. Here we show that amyloid-β oligomers bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. To determine the pathogenic importance of EphB2 depletions in Alzheimer’s disease and related models, we used lentiviral constructs to reduce or increase neuronal expression of EphB2 in memory centres of the mouse brain. In nontransgenic mice, knockdown of EphB2 mediated by short hairpin RNA reduced NMDA receptor currents and impaired long-term potentiation in the dentate gyrus, which are important for memory formation. Increasing EphB2 expression in the dentate gyrus of human amyloid precursor protein transgenic mice reversed deficits in NMDA receptor-dependent long-term potentiation and memory impairments. Thus, depletion of EphB2 is critical in amyloid-β-induced neuronal dysfunction. Increasing EphB2 levels or function could be beneficial in Alzheimer’s disease.

Phospholipase A2 reduction ameliorates cognitive deficits in a mouse model of Alzheimer's disease

Neuronal expression of familial Alzheimers disease–mutant human amyloid precursor protein (hAPP) and hAPP-derived amyloid-β (Aβ) peptides causes synaptic dysfunction, inflammation and abnormal cerebrovascular tone in transgenic mice. Fatty acids may be involved in these processes, but their contribution to Alzheimers disease pathogenesis is uncertain. We used a lipidomics approach to generate a broad profile of fatty acids in brain tissues of hAPP-expressing mice and found an increase in arachidonic acid and its metabolites, suggesting increased activity of the group IV isoform of phospholipase A2 (GIVA-PLA2). The levels of activated GIVA-PLA2 in the hippocampus were increased in individuals with Alzheimers disease and in hAPP mice. Aβ caused a dose-dependent increase in GIVA-PLA2 phosphorylation in neuronal cultures. Inhibition of GIVA-PLA2 diminished Aβ-induced neurotoxicity. Genetic ablation or reduction of GIVA-PLA2 protected hAPP mice against Aβ-dependent deficits in learning and memory, behavioral alterations and premature mortality. Inhibition of GIVA-PLA2 may be beneficial in the treatment and prevention of Alzheimers disease.

Neprilysin Overexpression Inhibits Plaque Formation But Fails to Reduce Pathogenic Aβ Oligomers and Associated Cognitive Deficits in Human Amyloid Precursor Protein Transgenic Mice

The accumulation of amyloid-β (Aβ) peptides in the brain of patients with Alzheimers disease (AD) may arise from an imbalance between Aβ production and clearance. Overexpression of the Aβ-degrading enzyme neprilysin in brains of human amyloid precursor protein (hAPP) transgenic mice decreases overall Aβ levels and amyloid plaque burdens. Because AD-related synaptic and cognitive deficits appear to be more closely related to Aβ oligomers than to plaques, it is important to determine whether increased neprilysin activity also diminishes the levels of pathogenic Aβ oligomers and related neuronal deficits in vivo. To address this question, we crossed hAPP transgenic mice with neprilysin transgenic mice and analyzed their offspring. Neprilysin overexpression reduced soluble Aβ levels by 50% and effectively prevented early Aβ deposition in the neocortex and hippocampus. However, it did not reduce levels of Aβ trimers and Aβ*56 or improve deficits in spatial learning and memory. The differential effect of neprilysin on plaques and oligomers suggests that neprilysin-dependent degradation of Aβ affects plaques more than oligomers and that these structures may form through distinct assembly mechanisms. Neprilysins inability to prevent learning and memory deficits in hAPP mice may be related to its inability to reduce pathogenic Aβ oligomers. Reduction of Aβ oligomers will likely be required for anti-Aβ treatments to improve cognitive functions.

The Disintegrin ADAM9 Indirectly Contributes to the Physiological Processing of Cellular Prion by Modulating ADAM10 Activity

The cellular prion protein (PrPc) is physiologically cleaved in the middle of its 106–126 amino acid neurotoxic region at the 110/111↓112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called “α-secretase pathway” that leads to the production of secreted sAPPα from βAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPα recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPα productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound α-secretase activity in ADAM10-/- fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10-/- cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrPc in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrPc and βAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106–126 and Aβ toxic domains of the prion and βAPP proteins.

Ablation of Cellular Prion Protein Does Not Ameliorate Abnormal Neural Network Activity or Cognitive Dysfunction in the J20 Line of Human Amyloid Precursor Protein Transgenic Mice

Previous studies suggested that the cellular prion protein (PrPc) plays a critical role in the pathogenesis of Alzheimers disease (AD). Specifically, amyloid-β (Aβ) oligomers were proposed to cause synaptic and cognitive dysfunction by binding to PrPc. To test this hypothesis, we crossed human amyloid precursor protein (hAPP) transgenic mice from line J20 onto a PrPc-deficient background. Ablation of PrPc did not prevent the premature mortality and abnormal neural network activity typically seen in hAPPJ20 mice. Furthermore, hAPPJ20 mice with or without PrPc expression showed comparably robust abnormalities in learning and memory and in other behavioral domains at 6–8 months of age. Notably, these abnormalities are not refractory to therapeutic manipulations in general: they can be effectively prevented by interventions that prevent Aβ-dependent neuronal dysfunction also in other lines of hAPP transgenic mice. Thus, at least in this model, PrPc is not an important mediator of Aβ-induced neurological impairments.

The C-terminal Products of Cellular Prion Protein Processing, C1 and C2, Exert Distinct Influence on p53-dependent Staurosporine-induced Caspase-3 Activation

The cellular prion protein (PrPc) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrPc yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrPc in cell survival.

M1 and M3 Muscarinic Receptors Control Physiological Processing of Cellular Prion by Modulating ADAM17 Phosphorylation and Activity

The cellular prion protein (PrPc) undergoes a physiological processing yielding the N-terminal fragment referred to as N1, the production of which can be constitutive or protein kinase C regulated. We show that activation of endogenous muscarinic receptors by carbachol and by the M1-selective agonist AF267B increases N1 recovery in an atropine-sensitive manner, in mouse embryonic primary neurons. To identify the muscarinic receptor subtype involved, we used human embryonic kidney HEK293 (HEK) cells stably overexpressing M1, M2, M3, or M4 receptor subtype. Carbachol and the selective M1 agonist AF267B dose dependently increased N1 release by HEK-M3 and HEK-M1 cells, respectively, whereas carbachol did not modify N1 production by HEK-M2 or HEK-M4 cells. We demonstrate that the increase of N1 was not attributable to modified trafficking to the membrane of either PrPc or the disintegrin metalloproteases ADAM10 or ADAM17. Furthermore, we establish that carbachol affects the overall phosphorylation of ADAM17 on its threonine and tyrosine but not serine residues, whereas levels of phosphorylated ADAM9 were not affected. Interestingly, carbachol also increases the hydrolysis of the fluorimetric substrate JMV2770, which mimicked the sequence encompassing the N1 site cleavage and was shown previously to behave as an ADAM protease substrate. Mutations of threonine 735 but not of tyrosine 702 of the ADAM17 cytoplasmic tail abolishes the carbachol-induced increase of N1, ADAM17 phosphorylation, and JMV2770-hydrolyzing activity in M1- and M3-expressing HEK293 cells. Thus, our data provide strong evidence that muscarinic receptor activation increases the physiological processing of PrPc by upregulating the phosphorylation state and activity of ADAM17 protease.

Alzheimer's disease: A prion protein connection

More than 20 million people worldwide have Alzheimers disease, yet its causes remain mostly uncertain. Fresh findings provide molecular clues, linking this disease to another neurodegenerative disorder.

Regulation of βAPP and PrPc Cleavage by α-Secretase: Mechanistic and Therapeutic Perspectives

Alzheimers disease (AD) is by far the most common form of dementia in the elderly and concerns one out of three individuals over 85. Like other neurodegenerative disorders such as Parkinson, Hungtington or prion diseases, AD is characterized by the formation of amyloid plaques in the central nervous system. In the brain of AD patients, the main component of these abnormal deposits is an aggregated form of the so-called amyloid beta-peptide (Abeta), which is produced from a large trans-membrane type-1 protein, the beta-amyloid precursor protein (betaAPP), by the sequential action of the beta- and gamma-secretases. Beside these two amyloidogenic proteolytic attacks, betaAPP is targeted by a third enzyme termed alpha-secretase. Of utmost importance, this cleavage, which can be of constitutive or regulated origin, occurs right in the middle of the Abeta sequence, thus precluding its production. For this reason, and because the sAPPalpha secreted fragment derived from this cleavage displays beneficial effects, tremendous efforts have been made recently in order to both identify the proteases involved and the way they are regulated. More recently, it emerged that alpha-secretase was also responsible for the physiological processing of the cellular prion protein (PrP(c)) in the middle of its toxic 106-126 sequence. This review will focus on the recent advances in the alpha-secretase pathways regulation and will discuss the putative therapeutic approaches that could be envisioned concerning the treatment of two apparently distinct diseases that share common denominators according to their metabolism.

The Extracellular Regulated Kinase-1 (ERK1) Controls Regulated α-Secretase-mediated Processing, Promoter Transactivation, and mRNA Levels of the Cellular Prion Protein

The α-secretases A disintegrin and metalloprotease 10 (ADAM10) and ADAM17 trigger constitutive and regulated processing of the cellular prion protein (PrPc) yielding N1 fragment. The latter depends on protein kinase C (PKC)-coupled M1/M3 muscarinic receptor activation and subsequent phosphorylation of ADAM17 on its intracytoplasmic threonine 735. Here we show that regulated PrPc processing and ADAM17 phosphorylation and activation are controlled by the extracellular-regulated kinase-1/MAP-ERK kinase (ERK1/MEK) cascade. Thus, reductions of ERK1 or MEK activities by dominant-negative analogs, pharmacological inhibition, or genetic ablation all impair N1 secretion, whereas constitutively active proteins increase N1 recovery in the conditioned medium. Interestingly, we also observed an ERK1-mediated enhanced expression of PrPc. We demonstrate that the ERK1-associated increase in PrPc promoter transactivation and mRNA levels involve transcription factor AP-1 as a downstream effector. Altogether, our data identify ERK1 as an important regulator of PrPc cellular homeostasis and indicate that this kinase exerts a dual control of PrPc levels through transcriptional and post-transcriptional mechanisms.