Alzheimer's disease (AD) is 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 β-amyloid peptide (Aβ) derived via proteolysis from the amyloid precursor protein APP. APP is a single pass transmembrane protein that is processed in two different ways: α-secretase cleaves APP within the Aβ region, thereby precluding Aβ formation and releasing the APPsα ectodomain; in the amyloidogenic pathway APP is sequentially cleaved by β- and ϒ-secretase, leading to Aβ 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.
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 β-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. Mice in which APP, APLP1, or APLP2 is inactivated are viable and APP-KO mice revealed 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 synaptic differentiation/function (Soba et al. 2005). Collectively, our data revealed an essential role for APP-family members in normal brain development and early postnatal survival. Work is in progress to circumvent early lethality and assess functions postnatally by generating tissue specific knockouts.
|Fig. 1 Frontal section of a triple KO mouse at E 17.5 exhibiting a prominent protrusion (P) of the cortical plate. Ectopic neurons completely disrupt the cortical plate (CP); and neuroblasts are shifted into the marginal zone (MZ). (adapted from Herms et al., 2004)|
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 relevance of APP processing and to understand the specific functions of the respective cleavage product for both physiology and pathophysiology. 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 (APPsα) or to a C-terminal truncation lacking the YENPTY interaction motif (APPΔCT15) (Ring et al., 2007). Interestingly, the ΔCT15-deletion resulted in reduced turnover of holoAPP, increased cell surface expression an 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 APP C-terminua is dispensable and that APPsα is sufficient to mediate the physiological functions of APP assessed by these tests (Ring et al., 2007). Ongoing experiments will show whether APPsα might also be sufficient to rescue defects underlying the lethal phenotype of APP-/-APLP2-/- mutants.
The role of APP and its fragments for neuronal morphology and synaptic function
Recently, a role of APP and APLP2 at the neuromuscular synapse has been described. APP/APLP2 double knockout mice showed a highly reduced frequency of miniature endplate potentials (MEPPs) associated with a reduction in synaptic vesicle density. Thus, ongoing work in the lab is directed to assess whether APP/APLP deficiency is also associated with related defects of neuronal morphology and/or synaptic function within the CNS. To this end we are analyzing organotypic hippocampal cultures of knockout and knockin mice with regard to morphology and (in collaboration) for their electrophysiological characteristics (basal synaptic transmission, synaptic plasticity).
Role of APP-dependent gene expression for Alzheimer disease
The proteolytical processing of APP is very similar to that of Notch and the APP intracellular domain 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/qPCR based approach we identified novel differentially expressed genes (e.g. involved in cytoskeletal remodeling, endocytosis, cell adhesion and neurotransmitter systems). In this context we are particularly interested to clarify whether these 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.
Regulation and molecular pathology of the inhibitory glycine receptor
Glycine receptors (GlyRs) are ligand activated chloride channels composed of α- and β subunits forming a pentamer. GlyRs are involved in the control of spinal motor and sensory pathways, but little was known about the biological roles of different GlyR subtypes. Recently we showed that GlyRα3 subunits are distinctly expressed in superficial laminae of the dorsal spinal cord, the first site of synaptic integration in the pain pathway (Harvey et al., 2004). At this site, glycinergic neurotransmission is inhibited by prostaglandin E2 (PGE2), a pivotal mediator of inflammatory pain sensitization. By generation of GlyRα3 knockout mice we could show that this receptor subtype plays a crucial role in spinal nociceptive processing. Mice deficient in GlyRα3 not only lacked the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice, but also showed a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation (Harvey et al., 2004). Thus, GlyRα3 may provide a novel molecular target in pain therapy.
|Fig. 2. Transverse section through wild type spinal cord. Double labeling shows that GlyRα3 (green) is restricted to the dorsal horn, and gephyrin (red) is expressed throughout the gray matter. (adapted from Harvey et al., 2004)|