Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons

SummaryAxon guidance relies on precise translation of extracellular signal gradients into local changes in cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here, we show that during embryonic development in growing axons, a low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A levels specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo, leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance

A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.American Heart Association (Fellowship 0615692T)National Institutes of Health (U.S.) (Grant GM68678

Impairment of Adolescent Hippocampal Plasticity in a Mouse Model for Alzheimer's Disease Precedes Disease Phenotype

The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in combination with DIGE and mass spectrometry. We investigated cortex and hippocampus of transgenic and wildtype mice at 1, 2, 7 and 15 months of age. Furthermore, cortices of 16 days old embryos were analyzed. When comparing the protein patterns of APP23 with wildtype mice, we detected a relatively large number of altered protein spots at all age stages and brain regions examined which largely preceded the occurrence of amyloid plaques. Interestingly, in hippocampus of adolescent, two-month old mice, a considerable peak in the number of protein changes was observed. Moreover, when protein patterns were compared longitudinally between age stages, we found that a large number of proteins were altered in wildtype mice. Those alterations were largely absent in hippocampus of APP23 mice at two months of age although not in other stages compared. Apparently, the large difference in the hippocampal protein patterns between two-month old APP23 and wildtype mice was caused by the absence of distinct developmental changes in the hippocampal proteome of APP23 mice. In summary, the absence of developmental proteome alterations as well as a down-regulation of proteins related to plasticity suggest the disturption of a normally occurring peak of hippocampal plasticity during adolescence in APP23 mice. Our findings are in line with the observation that AD is preceded by a clinically silent period of several years to decades. We also demonstrate that it is of utmost importance to analyze different brain regions and different age stages to obtain information about disease-causing mechanisms

The Intracellular Threonine of Amyloid Precursor Protein That Is Essential for Docking of Pin1 Is Dispensable for Developmental Function

Background: Processing of Ab-precursor protein (APP) plays an important role in Alzheimer’s Disease (AD) pathogenesis. Thr residue at amino acid 668 of the APP intracellular domain (AID) is highly conserved. When phosphorylated, this residue generates a binding site for Pin1. The interaction of APP with Pin1 has been involved in AD pathogenesis. Methodology/Principal Findings: To dissect the functions of this sequence in vivo, we created an APP knock-in allele, in which Thr 668 is replaced by an Ala (T 668 A). Doubly deficient APP/APP-like protein 2 (APLP2) mice present postnatal lethality and neuromuscular synapse defects. Previous work has shown that the APP intracellular domain is necessary for preventing early lethality and neuromuscular junctions (NMJ) defects. Crossing the T 668 A allele into the APLP2 knockout background showed that mutation of Thr 668 does not cause a defective phenotype. Notably, the T 668 A mutant APP is able to bind Mint1. Conclusions/Significance: Our results argue against an important role of the Thr 668 residue in the essential function of APP in developmental regulation. Furthermore, they indicate that phosphorylation at this residue is not functionally involved i

Die Trim2 Mausmutante - Ein Model für Ataxia des Zerebellums

Im Rahmen eines großangelegten Gene-Trap-Mutations-Screens mit dem Ziel, neue an der Hirnentwicklung und -funktion beteiligten Gene zu identifizieren, wurde eine Gene-Trap (GT) Maus-Linie charakterisiert, die eine postnatale Reportergen-Expression in zerebellaren Purkinje-Zellen (PZ), im Hippokampus, im Kortex und in der Ganglienzell- und inneren Körnerschicht der Retina aufweist. Die für diese Gen-Trap -Mutation homozygoten Mäuse waren bis zu einem Alter von ca. 1.5 Monaten phänotypisch nicht von ihren Wildtyp-Wurfgeschwistern unterscheidbar, wonach ein Intentionstremor gefolgt von einer Gangataxie aufzutreten begann. In späteren Stadien (um die drei Monate) waren spontane epileptische Anfälle zu beobachten. Histologisch litten die mutierten Mäuse an einer progressiven Degeneration von Purkinje-Zellen und tiefen zerebellären Kernen. Die Innervation der Purkinje-Zellen durch die Kletterfasern wurde bei den mutierten Mäusen zunächst ausgebildet, dann aber zurückgezogen und hatte eine Degeneration der Purkinje-Zellen zur Folge. In der Retina kam es zu einer Größenreduzierung in der Ganglienzell- und der inneren Körnerschicht. Die Größe der äußeren plexiformen Schicht, in der sich die Dendriten der Bipolarzellen befinden, war ebenfalls signifikant reduziert. Im Hippocampus fand sich keine Degeneration.Die GT Vektor-Insertion trat innerhalb einer RBCC/TRIM RING-finger Domäne auf, die das Gen trim2 enthält einen Bindungspartner von Myosin V. Myosin V ist, als ein Motorprotein, für den Transport des glatten endoplasmatischen Retikulums in die dendritischen Dornen der Purkinje-Zellen verantwortlich. Zudem interagiert es mit der leichten Untereinheit des Neurofilaments (NF-L) und reguliert die Neurofilament-Dichte in Axonen. Da die Dendriten in trim2 homozygoten Mäusen keinen Phänotyp aufwiesen, jedoch bereits in einerthalb-jährigen Mutanten axonale Anschwellungen sowohl in der zerebellaren weißen Substanz, als auch in der Körnerzellschicht auftraten (sogar noch vor der Degeneration der Purkinje-Zellen), nehmen wir an, daß der axonale Defekt eine Folge des zerebellären Phänotyps der trim2 Mausmutanten ist