2013

• Küffner, Mercedes
  Ultrastructural analysis of spine apparatus in CA3 pyramidal neurons following single cell electroporation in Synaptopodin Knockout - mice
  2013 Dissertation, Institut für Anatomie und Zellbiologie, Erstgutachter: Frotscher, Michael, Zweitgutachter: Aertsen, Ad
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  Abstract

  The spine apparatus, a membranous extension of the dendrititc smooth endoplasmic reticulum, is located in mature dendritic spines of cortical and hippocampal neurons. This neuronal organelle exists only in a subpopulation of neurons, in large mushroom-shaped spines. The telencephalic protein Synaptopodin obtains a critical role in the formation of spine apparatuses, as this organelle is absent in Synaptopodin-knockout mice. Synaptopodin has been shown to bind actin and actin polymerization in turn contributes to the maintainance of the spine morphology. The overall aim of this thesis was to reveal molecular mechanisms for morphological alterations of CA3 pyramidal neurons at the cellular and subcellular level. To achieve this, single-cell electroporation of CA3 pyramidal neurons in organotypic hippocampal slice cultures was chosen to manipulate the expression of the axonal cell adhesion molecule L1CAM and the postsynaptic protein synaptopodin. Morphology of CA3 pyramidal neurons was successfully modulated after electroporation of L1CAM and two L1CAM missense mutations. These mutations were shown to interfered with axonal targeting of L1CAM and L1CAM-dependent stimulation of axonal branching. Genetic complemetation of synaptopodin-deficient neurons lead to the de novo formation of spine apparatuses in a cell autonomous manner. The restoration process of spine apparatsues is exclusively mediated by synaptopodin, as transfection studies with KIBRA did not lead to formation of spine apparatuses in spines or dendrites. After genetic complementation the general morphology of these organelles was enlarged in mushroom-shaped spines and dendrites and the total number of spine apparatuses increased in parent dendrites. Pharmacological inhibition of small Rho-GTPases led to increased spine apparatuses in the wild-type, which can be traced back to depleted interdigitating F-actin filaments in between single tubules of the spine apparatus. Changes in spine apparatus morphology correlate with morphological changes in mushroom-shaped spines, as all analyzed conditions with a persistent increase in median surface area of spine apparatuses showed additionally a significant increase for appropriate spines.

2010

• Schäfer MK, Nam YC, Moumen A, Keglowich L, Bouche E, Küffner M, Bock HH, Rathjen FG, Raoul C, Frotscher M
  L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms.
  2010 Neurobiol Dis, volume: 40, issue: 1, pages: 222 - 237
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  Abstract

  Mutations in the human L1CAM gene cause neurodevelopmental disorders collectively referred to as L1 syndrome. Here, we investigated cellular pathomechanisms underlying two L1 syndrome mutations, R184Q and W1036L. We demonstrate that these mutations cause partial endoplasmic reticulum (ER) retention of L1, reduce L1 cell surface expression, but do not induce ER stress in neuronal NSC-34 cells. We provide evidence that surface trafficking of mutated L1 is affected by defective sorting to ER exit sites and attenuated ER export. However, in differentiated neuronal cultures and long-term cultured hippocampal slices, the L1-R184Q protein is restricted to cell bodies, whereas L1-W1036L also aberrantly localizes to dendrites. These trafficking defects preclude axonal targeting of L1, thereby affecting L1-mediated axon growth and arborization. Our results indicate that L1 syndrome mutations impair neuronal L1 function at different levels, firstly by attenuating ER export and secondly by interfering with polarized neuronal trafficking.