Regulation of Embryonic Cell Adhesion by the Prion Protein

Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development

mTORC2 controls neuron size and Purkinje cell morphology independent of mTORC1

Prenatal brain development is mainly accomplished by extensive proliferation of neuronal precursor cells whereas postnatal brain growth in mammals is mainly mediated by the growth of those post-mitotic nerve cells. The neuron size and the branching pattern of the dendritic tree are highly controlled during development to enable the proper connectivity of neuronal circuits and the accurate electrical transmission in the adult which is a prerequisite for the brain to function normally. Aberrations in size, morphology or connectivity have been shown to be the cause for various brain disorders. Neuron size and dendrite development are controlled by intrinsic mechanisms, trophic factors and neuronal activity, processes that need the concerted action of a plethora of signaling molecules. A central integrator of various signaling cascades is the mammalian target of rapamycin (mTOR) and as such it contributes to brain development and function and is thus also implicated in the pathophysiology of psychiatric disorders. mTOR is a serine threonine protein kinase that is highly conserved from yeast to humans and has been found to be part of at least two multi-protein complexes mTORC1 and mTORC2. The formation of mTORC1 is dependent on the protein raptor whereas mTORC2 assembly relies on the protein rictor. In recent years a complex picture about the function of mTORC1 has emerged by use of rapamycin, an immunosuppressive drug that acutely inhibits mTORC1 formation and activity and has attributed mTORC1 a major role in the regulation of cell size and proliferation. However, because the activity of mTORC2 is only depleted upon long term application of rapamycin, research advancement on its function was thus far impeded. Due to the early embryonic lethality of raptor or rictor knockout in mammals conditional knockout models were constructed. Whereas tissue specific knockout of raptor led to characteristic alterations, knockout of rictor in several organs such as skeletal muscle and adipose tissue provided none or only a weak phenotype. Several cell culture studies assigned mTORC2 a role in cytoskeletal modifications but in vivo confirmation is still lacking. The current knowledge about mTORC2 is restricted to the downstream targets Akt/PKB (proteinkinase B) and PKC (protein kinase C) which belong to the AGC kinase family. Those kinases are reported to influence cell morphology, growth and survival and are also essential regulators of brain development and function. PKCs are involved in synaptic plasticity and neurotransmitter release and, hence, also in the pathophysiological mechanisms of psychiatric disorders especially in schizophrenia and bipolar disorder. Concordantly, several psychiatric agents have been shown to alter PKC signaling. This emphasizes the urge to analyze the role of mTORC2 in the central nervous system. In this dissertation the role of mTORC2 was analyzed in the central nervous system and in specific sub-populations of neurons by deletion of rictor. I discovered, that in contrast to all other organs analyzed so far, rictor knockout in the brain reveals a pronounced phenotype. The brain-size of those mice shows an enormous reduction to almost half of that of control mice which is caused mainly by the reduction of neuron size. The reduced cell size is observed in neurons derived from different brain areas in vitro and in vivo but is most prominent in Purkinje cells of the cerebellum, the cell type with highest rictor expression. In addition, dendrite morphology is majorly disrupted and the formation of dendritic spines is affected which correlates with a decreased neuronal activity. The Purkinje cell phenotype can also be reproduced in a Purkinje cell specific knockout of rictor and thus demonstrates that the effect of rictor deletion in neurons is cell autonomous. Moreover, Purkinje cell axonal path-finding is affected which correlates with the decrease in phosphorylation of the neuron specific PKC target protein GAP-43, a known regulator for axon growth and path-finding. Molecular analysis reveals that rictor is essential for the activity of all conventional PKC isoforms and the novel PKCε in vivo and in vitro in neurons which influences the function of downstream targets important for cytoskeleton modifications such as GAP-43, MARCKs and neurofascin. In addition, rictor controls the phosphorylation of Akt but does not alter mTORC1 signaling towards its downstream effectors. In summary it becomes clear that rictor is important in the development and maturation of neurons and controls their size and neuron structure which influences the entire brain function and affects the behavior of the mice. Thus, these data encompass a new role of rictor in CNS disorders

Inactivation of mTORC1 in the Developing Brain Causes Microcephaly and Affects Gliogenesis

The mammalian target of rapamycin (mTOR) regulates cell growth in response to various intracellular and extracellular signals. It assembles into two multiprotein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTORC2. In this study, we inactivated mTORC1 in mice by deleting the gene encoding raptor in the progenitors of the developing CNS. Mice are born but never feed and die within a few hours. The brains deficient for raptor show a microcephaly starting at E17.5 that is the consequence of a reduced cell number and cell size. Changes in cell cycle length during late cortical development and increased cell death both contribute to the reduction in cell number. Neurospheres derived from raptor-deficient brains are smaller, and differentiation of neural progenitors into glia but not into neurons is inhibited. The differentiation defect is paralleled by decreased Stat3 signaling, which is a target of mTORC1 and has been implicated in gliogenesis. Together, our results show that postnatal survival, overall brain growth, and specific aspects of brain development critically depend on mTORC1 function

Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology

The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons