Current Research

Alzheimer’s disease (AD) is characterized by a gradual neuronal degeneration and loss of synapses. The microtubule associated protein Tau and the Amyloid Precursor Protein (APP) were identified as key players in the pathology of AD, but the reasons for the loss of synaptic connections and the sequential steps of neurodegeneration in the course of AD are not understood yet. The APP gene family consists in mammals of APP, APLP1 and APLP2, and we could show that it functions in diverse biological processes, such as cell adhesion, regulation of synaptic function and axonal transport. We assume that deregulation of the normal APP physiological function causes neurodegeneration and loss of synapses that might be causative for AD and possibly explain the high risk of aging in AD.

Kins Fig1
Fig.1: Modell of APP function at the synapse The members of the APP gene family, APP, APLP1 and APLP2, form homo- and heterotypic transdimers at the synapse. Further sAPP, promoting neuronal outgrowth, is secreted in the synaptic cleft. Both features argue for an importat role of APP/APLPs in synaptogenesis and/or synaptic plasticity



Specifically our research is addressing the following aims:

1. Determination of the molecular mechanisms underlying APP anterograde transport

APP is anterogradely transported by very high velocity (≤10 ?m/sec) and is possibly cleaved while transport along the axon to the presynaptic terminal. It has been reported that APP couples the motor machinery by direct binding to kinesin-1. However, recent work from our and several other research groups suggests that the hypothesis that APP serves as a kinesin-1 receptor and that the proteolytic processing machinery responsible for generating Aß is transported in the same vesicular compartment in axons of peripheral nerves requires revision (Back et al., 2007; Lazarov et al., 2005; Rusu et al., 2007). In a proteomic approach we now identified components of the active zone complex as a kinesin cargo receptor of APP transport vesicles, cotransporting α-secretase. As α-secretase activity abolish generation of the neurotoxic Aß peptide that accumulates in plaques of AD patients, we assume that changes in the velocity of APP anterograde transport, as described for aged neurons, play a pivotal role in the etiology of AD. We will determine the molecular composition of the content of the APP transport vesicle type, the precise mode of interaction of the motor machinery and the physiological consequences of alterations in transport velocity on APP pathogenic and physiological function.

2. Characterization of the cell adhesion features of APP/APLPs

We found that APP/APLPs can trigger cell-cell contacts via homo- and heterotypic transdimerization of the ectodomains (Soba et al., 2005). Interestingly, the hetero-complex of APP and APLP1 can be co-immunoprecipitated from synaptic membrane fractions, suggesting a putative function of APP/ APLPs transdimerization at synaptic sites. To extend these studies we started now detailed immunohistological analysis with newly generated anti-APP/APLPs antibodies on the ultra structural level of neuronal tissues and investigate in cell culture models its impact on synaptogenesis and synaptic plasticity. Further we initiated studies addressing the consequences of APP/APLPs dimerization on processing and cell migration. These analyses will give new insights in the physiological function of APP/APLPs and its relevance for neurodegeneration in AD.

 Kins Fig2
Fig. 2: Model for cis- and trans-interaction of APP family proteins The schematic model of APP/APLP domain organization and interaction is shown. The N-terminal E1 domain is linked to a highly flexible acidic region, followed by the alternatively spliced Kunitz-type protease inhibitor (KPI) domain (for APP and APLP2), the E2 domain, the juxta-/transmembrane (TM) region, and the cytosolic domain. Based on our results, we suggest that APP family proteins are capable of forming lateral and adhesive dimers in a homo- and heterotypic fashion. The E1 domain is crucial for both, cis- and trans-interaction, while the TM region could additionally contribute to lateral dimerization.

3. Investigtions of the amyloidogenic pathway of APP processing in recycling vesicles

For the understanding of intracellular transport it is of high impact to determine crucial sorting motifs and the underlying molecular machinery, including scaffolding, regulatory and motor proteins. We found that PAT1a binds to the basolateral sorting signal of APP/APLPs and affects theirs subcellular localization and cleavage by α- and ß-secretase (Back et al., 2007; Kuan et al., 2006). We want to determine the molecular mechanisms underlying the effect of PAT1a on APP/APLPs transport and processing. For this purpose we perform structural analysis and investigate the influence of PAT1a on the sorting of APP/APLPs in the endocytotic pathway in neuronal cells. Using the yeast twohybrid system, we identified a novel Rab5GEF that regulates Rab5-dependent endocytosis as a novel interaction partner of PAT1a. Currently, we determine the molecular interplay of Rab5, the novel identified Rab5GEF, PAT1a and APP/APLPs in the endocytotic pathway of APP/APLPs.

Kins Fig3
Fig. 3 Neuronal sorting of APP to dendrites and axons. Fluorescence microscopy image of a primary mouse neurons (DIV8) overexpressing mutant APP co-stained with anti-myc (green) and anti-MAP2 (red) antibodies. Arrowheads mark dendrites, arrows mark axons. Rectangular fields represent areas of quantification. For details see Back et al., 2007.

4. In vivo analyses of APP /APLPs neuronal function using viral miRNA

Investigations of the loss of function of APP/APLPs in adult mice were prohibited by early postnatal death of the doubleand triple knock-out mice. We have generated lentiviral constructs driving miRNA and EGFP mRNA expression under a Type II polymerase promoter. After unilateral stereotactic injection of the recombinant virus in different regions of the adult brain, we will analyse those neurons with reduced protein levels of APP, APLP1 or APLP2. The knock-down cells will be analyzed by immunhistological, electronmicroscopic and electrophysiological methods.

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Latest Revision: 2012-08-30
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