|Place of birth||München, Germany|
|Date of birth||10.04.1960|
|since 2005||Professor for Funktional Genomik, Institute for Pharmacy and molecular biotechnology, University of Heidelberg|
|1999||Habilitation in Molecularbiology, University of Zürich|
|1997-2004||Leader of the independent Neurogenetics research group (C3 for 5 years)|
|1991-1997||EMBO longterm fellow and independent research group leader, Institute for Molecular Biology, University of Zürich|
|1989-1991||Postdoc Medical School, University of Manchester, UK|
|1989||Dipl. rer. nat. Biochemistry and Molecular Biology, University of Munich|
|1985||Dipl. Chemistry, Universiy of Munich|
1. Background and Introduction
Alzheimer´s disease (AD) ist the most common neurodegenerative disorder and is characterized by synaptic disfunction, neuronal loss and cognitive decline. The major lesions found in the brains of AD patients are neruofibrillary tangles and neuritic plaques that are mainly composed of the b-amyloid peptide (Ab) derived via proteolysis from the amyloid precursor protein APP. APP is a single pass transmembrane protein that is processed in two different ways: a-secretase cleaves APP within the Ab region, thereby precluding Ab formation and releasing the APPsa ectodomain; in the amyloidogenic pathway APP is sequentially cleaved by b- and g-secretase, leading to Ab formation.
Whereas the mechanisms governing Aβ generation have been intensely studied, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown.
1.1 Knockout mice with individual or combined gene deficiencies of APP-family proteins
Determining the in vivo functions of APP in mammals is complicated by the presence of two APP-related genes, APLP1 and APLP2. APP and APLPs share two conserved domains in the extracellular region (E1 and E2) and one in the cytoplasmic domain, whereas the the b-amyloid peptide is lacking in APLPs.
Thus, functional redundancy may compensate for the loss of essential gene functions, e.g. in knockout (KO) models. Indeed, by generating various KO mutants we could demonstrate that the extensive structural similarities between APP and APLPs are also reflected at the functional level (Anliker and Müller, 2006). Mice in which APP, APLP1, or APLP2 is inactivated are viable and APP KO mice revelaed reduced brain and body weight, reduced grip strength, altered locomotor activity, increased susceptibility to seizures and a defect in spatial learning and LTP.
In contrast to the viable single mutants, combined APLP2-/-APP-/- and APLP2-/-APLP1-/- double mutants die shortly after birth indicating that APP family proteins serve redundant functions that are essential for viability (Heber et al., 2000). Whereas the brains of double knockout animals exhibit no obvious morphological defects, triple mutants lacking the entire APP gene family showed cranial dyspalsias resembling human type II lissencephaly (Herms et al., 2004).
Within affected areas neuronal cells from the cortical plate migrated beyond their normal positions and protruded into the marginal zone and the subarachnoid space. Thus, APP/APLPs play a critical role in neuronal adhesion and positioning. This role in cell adhesion is also supported by data from a recent collaborative study demonstrating that APP family proteins form cis- and trans-dimers involved in cellular adhesion and may thus play a role in neuronal differentiation, synapse formation and function (Soba et al. 2005). Collectively, our data reveal an essential role for APP-family members in normal brain development and early postnatal survival.
Fig1: Frontal section of a triple KO mouse at E 17.5 exhibiting a prominent protrusion (P) of the cortical plate. It becomes apparent that ectopic neurons completely disrupt the cortical plate (CP); the neuro- blasts are shifted into the marginal zone (MZ). (adapted from Herms et al., 2004)
1.2 In vivo analysis of APP functional domains
Secondly, another level of functional diversity may result from the complex proteolytic processing of APP and its APLP homogues by several secretases leading to diverse extra- and intracellular APP/APLP fragments. As these secretases have become major targets of therapeutic intervention it is of primary importance to elucidate the physiological function(s) of APP/APLPs and their processing products, because alterations in the activity or concentration of these fragments might have physiological consequences in itself.
Ultimately we would like to elucidate the physiological relevance of APP processing and to understand the specific functions of the respective cleavage product for both physiology and pathophysiolog. To this end we recently started a reverse genetic analysis of APP functional domains. We replaced the endogenous APP locus by gene targeted alleles and generated two lines of knockin mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPsa) or to a C-terminal truncation lacking the YENPTY interaction motif (APPDCT15) (Ring et al., 2007). Interestingly, the DCT15-deletion resulted in reduced turnover of holoAPP, increased cell surface expression and largely reduced Aβ levels in brain, likely due to reduced processing in the endocytic pathway.
Most importantly, we demonstrated that in both APP knockin lines the expression of APP N-terminal domains either largely attenuated or completely rescued the prominent deficits of APP knockout mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and LTP. Taken together our data suggest that the APP C-terminus is dispensable and that APPsa is sufficient to mediate the physiological functions of APP assessed by these tests (Ring et al., 2007). Ongoing experiments will show whether APPsa might also be sufficient to rescue defects underlying the lethal phenotype of APP-/-APLP2-/- mutants.
2. Outline of planned projects
The prize will be used to support our ongoing and planned efforts aimed at understanding the physiological as well as the pathological role of APP family proteins for Alzheimer´s disease and aging. To this end we intend to answer the following questions:
· What are the gene specific functions of the individual family members within the nervous systems, in particular with regard to neuronal differentiation, synaptogenesis, synaptic function and plasticity, as well as for learning and memory?
· In as much are these functions redundant within the gene family?
· What is the physiological function of the different proteolytic fragments generated from APP/APLPs (holoAPP, APPs, AICD)?
· What is the specific role of these fragments for AD pathogenesis?
· Can we do structure/function analysis?
· Which signaling mechanisms are involved and which genetic and molecular interactions are mediating these functions?
To this end we intend to use a combination of genetic and biochemical/cell biological approaches:
2.1 Reverse genetics of APP function in mice: role of APP and its fragments in the developing and adult nervous system.
Our recent paper by Ring et al. (2007) suggested that APPsa seems to be sufficient to mediate the (postnatal) functions of APP (at least as assessed by the various tests used). It is also clear, however, that APLP2 (which is still expressed at normal levels in KI mice) might have masked functional deficits, that can only be fully assessed on an APLP2-deficient background.
In this regard it will be crucial to determine whether APPsa is also sufficient to rescue the perinatal lethality of APLP2-/-APP-/- double knockout (DKO) mice. We have therefore crossed both APP-KI mutant with APLP2-/-mice and generated APPsα-KI/APLP2-/- and APP∆CT-KI/APLP2-/- double knockin (DKI) mice. So far, only very few litters have been analyzed, but preliminary analysis showed that DKI mice are viable, have grossly reduced body weight and exhibit abnormal motor behaviors. To phenotype these mice we will conduct a detailed anatomical/morphological characterization of the brain cytoarchitecture, determine neuronal number and morphology, with particular emphasis on CNS structures prominently affected in Alzheimers disease, such as hippocampus and cortex. As APP/APLPs have been implicated in synaptogenesis at the neuromuscular junction as well as in the CNS we will study in adult mice the synaptic architecture within the hippocampus. In addition we will conduct in colaboration an electrophysiological analysis of CNS synapses (e.g. in cortex and hippocampus) and assess potential deficits in short term and long term synaptic plasticity.
These studies will be complemented by an in depth analysis of neuronal morphology (dendritic complexity, synapse number, spine density) of principal neurons of the hippocampus in hippocampal slice cultures from our various gene targeted mice. Moreover, we will conduct a detailed behavioral characterization focusing on motor behaviours, sensory behaviours, as well as learning and memory. The second focus will be to extend our analysis of APP functional domains and generate additional gene targeted mouse mutants to study other important functional motifs including APPsb and the highly conserved E1 and E2 domains.
2.2 Generation of tissue-specific and inducible knockout mice.
Secondly, we intend to rescue the early postnatal lethality of the respective double mutants, in an effort to study functions at later stages of development and in the adult animal. Presently, the cause of the early postnatal phenotype of combined APP/APLP double and triple mutants is still unknown, raising the question in which organ system(s) the major functional defect may reside. To clarify this issue it will be crucial to generate APP/APLP single and combined gene deficiencies in a tissue specific, e.g. neuron specific way. Using tissue specific and tamoxifen-inducible cre-mediated deletion of the APP (or APLP2) gene we intend to analyse proposed functions in different neuronal populations during development, postnatally and in the adult.
2.3 Role of APP-dependent gene expression for Alzheimer disease
The proteolytical processing of APP is very similar to that of Notch and AICD has been suggested to function as a transcriptional regulator. Nevertheless, the nature of the relevant target genes is still under debate (see e.g. Pardossi-Picard et al., 2005 and Hebert et al., 2006). Using a microarray based approach we recently identified differentially expressed genes using cells (e.g. APP/APLP-deficient versus APP-reconstituted MEFs) and tissue (e.g. cortex and hippocampus) derived from single and combined APP/APLP mutants.
Thus, we identified several molecular pathways in which APP/APLPs are involved. Current work is aimed at follow up analysis of several interesting candidate genes (including e.g. neurotransmitter receptors , cell adhesion molecules). In this context we are particularly interested to clarify whether these new target genes are directly regulated at the mRNA level via AICD acting in a Notch-like manner, or are more indirectly affected by the absence of APP-family proteins.
2.5 Identification of extracellular and intracellular interaction partners
APP has since long been hypothesized to function as a cell surface receptor and to transduce various signals and so far only few binding partners have been identified. However, the in vivo ligands of APP/APLPs have remained unknown. We therefore intend to identify and functionally characterize such ligands using various interaction screens such as proteomics, pull down, immunoprecipitation and memebrane-based yeast two-hybrid screens.
Anliker, B. and Müller, U. (2006). The functions of the mammalian amyloid precursor protein and related amyloid precursor-like proteins. Neurodegenerative Diseases 3, 239-246.
Caille, I., Alliquant, B., Dupont, E., Bouillot, C., Langer, A., Müller, U. and Pronchiantz, A. (2004). Soluble form of Amyloid precursor protein regulates proliferation of Progenitors in the adult subventricular zone. Development, 131, 2173.
Grimm MO, Grimm HS, Patzold AJ, Zinser EG, Halonen R, Duering M, Tschape JA, De Strooper B, Muller U, Shen J, Hartmann T (2005) Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin. Nat Cell Biol, 7,1118-1123.
Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A., Rülicke, T., Kretzschmar, H., von Koch, C., Sisodia, S., Tremml, P., Lipp, H.-P., Wolfer, D. P. and Müller, U. (2000). Mice with combined gene knockouts reveal essential and partially redundant functions of Amyloid precursor protein family members. Journal of Neuroscience 20, 7951-7963.
Hebert SS, Serneels L, Tolia A, Craessaerts K, Derks C, Filippov MA, Muller U, De Strooper B (2006) Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes. EMBO Rep, 7,739-745.
Herms, J., Anliker, B., Heber, S., Ring. S., Fuhrmann, M., Kretzschmar, H., Sisodia, S. and Müller, U. (2004). Cortical Dysplasia Resembling Human Type 2 Lissencephaly in Mice Lacking all Three APP-Family Members. The EMBO J. 23, 4106 – 4115
Pardossi-Piquard, R. Petit, A., Kawarai, T., Sunyach, C. Alvers da Costa, C. Vincent, B., St. Ring, S. , DÁdamio, L., Shen, J., Müller, U., George Hyslop, P. and Checler, F. (2005). Presenilin-dependent transcriptional control of the Ab degrading enzyme neprilysin by intracellular domains of bAPP and APLP. Neuron, 46, 541-554
Ring. S, Weyer, S., Kilian, S. B., Waldron, E., Pietrzik, C. U., Filippov, M., Herms, J., Buchholz, C., Eckman, C. B., Martin Korte, M., Wolfer, D. P. and Müller, U. (2007) The secreted APPsα domain is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP deficient mice J. Neuroscience, 27, 7817-7826
Soba, P. , Eggert, S., Zentgraf, H., Siehl, K., Kreger, S., Löwer,A., Langer, A., Merdes, G., Paro, R.,. Masters, C. L., Müller,U., Kins, S. and Konrad Beyreuther. (2005) Homo- and hetero-dimerization of APP family members promotes intercellular adhesion. EMBO J., 24, 3624-3634