Fabrice Raynaud

Implication of calpain in neuronal apoptosis. A possible regulation of Alzheimer's disease.

Apoptotic neuronal cell death is the cardinal feature of aging and neurodegenerative diseases, but its mechanisms remain obscure. Caspases, members of the cysteine protease family, are known to be critical effectors in central nervous system cellular apoptosis. More recently, the calcium‐dependent proteases, calpains, have been implicated in cellular apoptotic processes. Indeed, several members of the Bcl‐2 family of cell death regulators, nuclear transcription factors (p53) and caspases themselves are processed by calpains. Progressive regional loss of neurons underlies the irreversible pathogenesis of various neurodegenerative diseases such as Alzheimers disease in adult brain. Alzheimers disease is characterized by extracellular plaques of amyloid–β peptide aggregates and intracellular neurofibrillary tangles composed of hyperphosphorylated tau leading to apoptotic cell death. In this review, we summarize the arguments showing that calpains modulate processes that govern the function and metabolism of these two key proteins in the pathogenesis of Alzheimers disease. To conclude, this article reviews our understanding of calpain‐dependent apoptotic neuronal cell death and the ability of these proteases to regulate intracellular signaling pathways leading to chronic neurodegenerative disorders such as Alzheimers disease. Further research on these calpain‐dependent mechanisms which promote or prevent cell apoptosis should help us to develop new approaches for preventing and treating neurodegenerative disorders.

Direct Interaction Enables Cross-talk between Ionotropic and Group I Metabotropic Glutamate Receptors

Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.

Alpha actinin–CapZ, an anchoring complex for thin filaments in Z-line

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and α-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for α-actinin, estimated by fluorescence and immunochemical assays, is in the μ m range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the α-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/α-actinin/titin/actin interactions is proposed considering Luthers 3D Z-line reconstruction.

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium‐dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097–2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z‐band and at the midpoint of the I‐band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I‐band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N‐terminal Z8–I5 region and the N1‐line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z‐band edge and the N1‐line as well as at the N2‐line level, two sarcomeric regions where early postmortem proteolysis is detected.

NF-κB-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A

Limb‐girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF‐NF‐κBB/INF‐κBBα survival pathway. In this study, the consequences of altered NF‐BNF‐κBB/IBNF‐κBBBα pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular‐FLICE inhibitory protein (C‐FLIP), which is dependent on the NF‐BNF‐κBB pathway in normal muscle cells, is down‐regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF‐BNF‐κBB is readily acti vated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF‐BNF‐κBB target genes. IBNF‐κBBBα is expressed following NF‐BNF‐κBB activation independent of the CAPN3 status, whereas expression of C‐FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF‐BNF‐κBB‐dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF‐BNF‐κBB pathway could be part of the mecha nism responsible for the muscle wasting resulting from CAPN3 deficiency.—Benayoun, B., Baghdiguian, S., Lajmanovich, A., Bartoli, M., Daniele, N., Gicquel, E., Bourg, N., Raynaud, F., Pasquier, M.‐A., Suel, L., Lochmuller, H., Lefranc, G., Richard, I. NF‐BNF‐κBB‐dependent expression of the antiapoptotic factor C‐FLIP is regulated by calpain 3, the protein involved in limb‐girdle muscular dystrophy type 2A. FASEB J. 22, 1521–1529 (2008)

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.

Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth.

The serotonin6 receptor (5-HT6R) is a promising target for treating cognitive deficits of schizophrenia often linked to alterations of neuronal development. This receptor controls neurodevelopmental processes, but the signaling mechanisms involved remain poorly understood. Using a proteomic strategy, we show that 5-HT6Rs constitutively interact with cyclin-dependent kinase 5 (Cdk5). Expression of 5-HT6Rs in NG108-15 cells induced neurite growth and expression of voltage-gated Ca(2+) channels, two hallmarks of neuronal differentiation. 5-HT6R-elicited neurite growth was agonist independent and prevented by the 5-HT6R antagonist SB258585, which behaved as an inverse agonist. Moreover, it required receptor phosphorylation at Ser350 by Cdk5 and Cdc42 activity. Supporting a role of native 5-HT6Rs in neuronal differentiation, neurite growth of primary neurons was reduced by SB258585, by silencing 5-HT6R expression or by mutating Ser350 into alanine. These results reveal a functional interplay between Cdk5 and a G protein-coupled receptor to control neuronal differentiation.

Shank3-Rich2 Interaction Regulates AMPA Receptor Recycling and Synaptic Long-Term Potentiation

Synaptic long-term potentiation (LTP) is a key mechanism involved in learning and memory, and its alteration is associated with mental disorders. Shank3 is a major postsynaptic scaffolding protein that orchestrates dendritic spine morphogenesis, and mutations of this protein lead to mental retardation and autism spectrum disorders. In the present study we investigated the role of a new Shank3-associated protein in LTP. We identified the Rho-GAP interacting CIP4 homolog 2 (Rich2) as a new Shank3 partner by proteomic screen. Using single-cell bioluminescence resonance energy transfer microscopy, we found that Rich2-Shank3 interaction is increased in dendritic spines of mouse cultured hippocampal neurons during LTP. We further characterized Rich2 as an endosomal recycling protein that controls AMPA receptor GluA1 subunit exocytosis and spine morphology. Knock-down of Rich2 with siRNA, or disruption of the Rich2-Shank3 complex using an interfering mimetic peptide, inhibited the dendritic spine enlargement and the increase in GluA1 subunit exocytosis typical of LTP. These results identify Rich2-Shank3 as a new postsynaptic protein complex involved in synaptic plasticity.

Binding of a native titin fragment to actin is regulated by PIP2

Titin is a giant protein which extends from Z‐line to M‐line in striated muscles. We report here the purification of a 150‐kDa titin fragment, obtained after V8 protease treatment of myofibrils. This polypeptide was located at the N1‐line level, in a titin part known to exhibit stiff properties correlated to an association with actin. By solid or liquid phase binding assays and cosedimentation, we have clearly demonstrated a direct, saturable and relative high affinity binding of the native titin fragment to F‐actin. The 150‐kDa titin fragment was also shown to accelerate actin polymerization. Furthermore, the actin‐titin interaction was found to be inhibited by phosphoinositides.

A Mouse Model for Monitoring Calpain Activity under Physiological and Pathological Conditions

Calpains are Ca2+-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the α-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.