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Titel: The roles of neural cell adhesion molecules NCAM and CHL1 in the regulation of synaptic ultrastructure in mice (Mus musculus Linnaeus, 1758)
Sonstige Titel: Die Rolle der neuralen Zelladhäsionsmoleküle NCAM und CHL1 in der Regulation von synaptischer Ultrastruktur bei Mäusen (Mus musculus Linnaeus, 1758)
Sprache: Englisch
Autor*in: Puchkov, Dmytro
Schlagwörter: Perforierte Synapsen; NCAM; Spectrin; Endocytosis; AMPA Receptoren; perforated synapses; NCAM; spectrin; endocytosis; AMPA receptors
GND-Schlagwörter: Perforierte Synapsen
NCAM
Spectrin
Endocytosis
AMPA Receptoren
Erscheinungsdatum: 2008
Tag der mündlichen Prüfung: 2008-05-23
Zusammenfassung: 
Project 1. The role of NCAM in maintaining of the cytoskeleton-dependent structural
integrity of post-synaptic densities and regulation of the AMPA receptor recycling

Glutamatergic synapses with postsynaptic densities (PSD) of complex shape, often referred as perforated, have attracted attention due to the transient increase of their percentage following neural activity, such as induction of long-term potentiation. Moreover, they are often considered as morphological landmarks of synaptic remodelling
and increased synapse activation in brain pathologies. In spite of the fact that models of PSD perforation have been widely discussed, mechanisms inducing perforation of PSDs and the role of this structural rearrangement in PSD function are poorly understood. Cell adhesion molecules and associated cytoskeletal elements are likely to play an important role in such rearrangements. The neural cell adhesion molecule NCAM has been implicated in synaptic plasticity and recruits its binding partner spectrin to synaptic contacts providing an anchoring scaffold for synaptic proteins. We show that numbers of perforated synapses are increased in the CA1 stratum radiatum of the hippocampus of NCAM deficient (NCAM-/-) versus wild type (NCAM+/+) mice and in cultured NCAM-/- versus NCAM+/+ hippocampal neurons. Disruption of the spectrin meshwork in cultured NCAM+/+ hippocampal neurons by antimycin or latrunculin or by transfection with spectrin siRNA increases the percentage of perforated synapses in these neurons to the level seen in NCAM-/- neurons, suggesting that the NCAM-assembled spectrin cytoskeleton maintains structural integrity of PSD. We demonstrate that PSD perforations
contain endocytic zones involved in alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalisation. AMPA receptor internalisation in NCAM-/- neurons is increased by 30% compared to NCAM+/+ neurons indicating that the NCAM assembled postsynaptic spectrin scaffold is important for inhibition of AMPA receptor endocytosis. An abnormally high AMPA receptor endocytosis being accompanied by structural abnormalities in PSD may contribute to alterations in synaptic plasticity and brain pathologies associated with ablation or mutations in NCAM and spectrin genes.


Project 2. The role of the cell adhesion molecule close homologue of L1 (CHL1) in
regulation of clathrin-dependent synaptic vesicle recycling

Mutations in human analogue of murin CHL1 gene – CALL correlate with the occurrence of schizophrenia. Schizophrenia is a neuropsychiatric disorder associated with
abnormal neurocircuits and functioning of synapses. Although these findings suggest that
CHL1 regulates synapse functioning, the role of CHL1 in the organization of the synaptic
machinery has not been fully analysed. Pilot experiments show that CHL1 interacts with 70 kDa heat shock cognate protein (Hsc70) that is a constitutively expressed member of the heat shock inducible Hsp70 protein family. Hsc70 regulates uncoating of clathrin coated vesicles in the clathrin-dependent synaptic vesicles recycling pathway. In the present study, we were able to show that CHL1 localizes in axons where it recruits Hsc70 to the synaptic terminals. Numbers of clathrin coated vesicles were increased in synaptic terminals of CHL1-/- neurons suggesting that clathrin uncoating is slowed down under conditions of reduced levels of Hsc70. Conversely, activity-induced formation of clathrin-coated vesicles in response to high potassium stimulation was inhibited in CHL1-/- compared with CHL1+/+ synapses. This can suggest that reduced rates of clathrin coat release from CCSVs may result in lack of availability of essential coat proteins to form new CCSVs in an activity-dependent manner: clathrin, AP180, and AP2
could be trapped in CCSVs formed during previous cycles of neuronal activity, and would thus be unavailable to form new CCSVs. Hsc70 is still recruited into the synapses of CHL1-/- neurons probably via CSP, another vesicle Hsc70 binding protein. Levels of this protein are elevated in CHL1-/- mice. To overcome this compensatory mechanism we have loaded CHL1+/+ neurons with a HPD peptide, which competitively interferes with CHL1/Hsc70 interaction. Disruption of CHL1/Hsc70 interaction showed an even more pronounced effect than CHL1 deficiency itself. HPD peptide induced two-fold increase in the numbers of clathrin coated vesicles and 20% decrease in the number of synaptic vesicles in non-stimulated synapses, nearly completely blocked activity-induced CCSV formation and significantly inhibited activity-induced synaptic vesicle fusion. Our results demonstrate that deficiency in CHL1/Hsc70 interaction inhibits uncoating of CCSVs and thus slower processing of synaptic vesicles recycling. This can contribute to synapse disregulations contributing to a set of neuropsychiatric disorder including schizophrenia.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/2205
URN: urn:nbn:de:gbv:18-37726
Dokumenttyp: Dissertation
Betreuer*in: Schachner, Melitta (Prof. Dr.)
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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