Another Use for a Proven Drug: Experimental Evidence for the Potential of Artemisinin and Its Derivatives to Treat Alzheimer's Disease.

Plant-derived multitarget compounds may represent a promising therapeutic strategy for multifactorial diseases, such as Alzheimer's disease (AD). Artemisinin and its derivatives were indicated to beneficially modulate various aspects of AD pathology in different AD animal models through the regulation of a wide range of different cellular processes, such as energy homeostasis, apoptosis, proliferation and inflammatory pathways. In this review, we aimed to provide an up-to-date overview of the experimental evidence documenting the neuroprotective activities of artemi-sinins to underscore the potential of these already-approved drugs for treating AD also in humans and propose their consideration for carefully designed clinical trials. In particular, the benefits to the main pathological hallmarks and events in the pathological cascade throughout AD development in different animal models of AD are summarized. Moreover, dose- and context-dependent effects of artemisinins are noted.

Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate.

Alzheimer's disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Brothers in arms: proBDNF/BDNF and sAPPα/Aß-signaling and their common interplay with ADAM10, TrkB, p75NTR, sortilin, and sorLA in the progression of Alzheimer's disease.

Brain-derived neurotrophic factor (BDNF) is an important modulator for a variety of functions in the central nervous system (CNS). A wealth of evidence, such as reduced mRNA and protein level in the brain, cerebrospinal fluid (CSF), and blood samples of Alzheimer's disease (AD) patients implicates a crucial role of BDNF in the progression of this disease. Especially, processing and subcellular localization of BDNF and its receptors TrkB and p75 are critical determinants for survival and death in neuronal cells. Similarly, the amyloid precursor protein (APP), a key player in Alzheimer's disease, and its cleavage fragments sAPPα and Aß are known for their respective roles in neuroprotection and neuronal death. Common features of APP- and BDNF-signaling indicate a causal relationship in their mode of action. However, the interconnections of APP- and BDNF-signaling are not well understood. Therefore, we here discuss dimerization properties, localization, processing by α- and γ-secretase, relevance of the common interaction partners TrkB, p75, sorLA, and sortilin as well as shared signaling pathways of BDNF and sAPPα.

The role of mycotoxins in neurodegenerative diseases: current state of the art and future perspectives of research.

Mycotoxins are fungal metabolites that can cause various diseases in humans and animals. The adverse health effects of mycotoxins such as liver failure, immune deficiency, and cancer are well-described. However, growing evidence suggests an additional link between these fungal metabolites and neurodegenerative diseases. Despite the wealth of these initial reports, reliable conclusions are still constrained by limited access to human patients and availability of suitable cell or animal model systems. This review summarizes knowledge on mycotoxins associated with neurodegenerative diseases and the assumed underlying pathophysiological mechanisms. The limitations of the common in vivo and in vitro experiments to identify the role of mycotoxins in neurotoxicity and thereby in neurodegenerative diseases are elucidated and possible future perspectives to further evolve this research field are presented.

Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density.

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer's disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.

Artesunate restores the levels of inhibitory synapse proteins and reduces amyloid-ß and C-terminal fragments (CTFs) of the amyloid precursor protein in an AD-mouse model.

Alzheimer's disease (AD) is the most frequent form of dementia, characterized histopathologically by the formation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid ß-peptide (Aß) is a major component of amyloid plaques and is released together with carboxy-terminal fragments (CTFs) from the amyloid precursor protein (APP) through proteolytic cleavage, thought to contribute to synapse dysfunction and loss along the progression of AD. Artemisinins, primarily antimalarial drugs, reduce neuroinflammation and improve cognitive capabilities in mouse models of AD. Furthermore, artemisinins were demonstrated to target gephyrin, the main scaffold protein of inhibitory synapses and modulate GABAergic neurotransmission in vitro. Previously, we reported a robust decrease of inhibitory synapse proteins in the hippocampus of 12-month-old double transgenic APP-PS1 mice which overexpress in addition to the Swedish mutated form of the human APP a mutated presenilin 1 (PS1) gene and are characterized by a high plaque load at this age. Here, we provide in vivo evidence that treating these mice with artemisinin or its semisynthetic derivative artesunate in two different doses (10 mg/kg and 100 mg/kg), these compounds affect differently inhibitory synapse components, amyloid plaque load and APP-processing. Immunofluorescence microscopy demonstrated the rescue of gephyrin and γ2-GABAA-receptor protein levels in the brain of treated mice with both, artemisinin and artesunate, most efficiently with a low dose of artesunate. Remarkably, artemisinin reduced only in low dose the amyloid plaque load correlating with lower levels of mutated human APP (hAPPswe) whereas artesunate treatment in both doses resulted in significantly lower plaque numbers. Correspondingly, the level of APP-cleavage products, specifically the amount of CTFs in hippocampus homogenates was reduced significantly only by artesunate, in line with the findings in hAPPswe expressing cultured hippocampal neurons evidencing a concentration-dependent inhibition of CTF-release by artesunate already in the nanomolar range. Thus, our data support artemisinins as neuroprotective multi-target drugs, exhibiting a potent anti-amyloidogenic activity and reinforcing key proteins of inhibitory synapses.

The Rab5 activator RME-6 is required for amyloid precursor protein endocytosis depending on the YTSI motif.

Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.

The amyloid precursor protein affects glyceraldehyde 3-phosphate dehydrogenase levels, organelle localisation and thermal stability.

Glyceraldehyde 3-phosphate dehydrogenase's (GAPDH) proapoptotic response to cellular oxidative stress has suspected implication for Alzheimer's disease (AD). Interestingly, the overexpression of the amyloid precursor protein (APP) can initiate oxidative stress responses within mammalian cell lines. Here, APP695 and APP770 overexpression significantly increased the level of GAPDH, while no effect was observed when the APP homologues APLP1 or APLP2 were used. Heterologous expression of APP695 was shown to increase the level of GAPDH within the cytoplasm by over 100% and within the mitochondria by approximately 50%. Moreover, a shift in organelle distribution from cytoplasm > nucleus > mitochondria in control cell lines to cytoplasm > mitochondria > nucleus in the APP695 overexpressing cell line was also observed. Further, the overexpression of APP695 increased GAPDH aggregation temperature by 3.09 ± 0.46 °C, indicative of greater thermal stability. These results demonstrate a clear correlation between APP overexpression and GAPDH levels, organelle distribution and thermal stability.

Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein 1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growth factor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and in vitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

Amyloidosis causes downregulation of SorLA, SorCS1 and SorCS3 expression in mice.

Accumulation of ß-amyloid peptide (Aß) is regarded as a primary cause of Alzheimer's disease (AD). Aß is derived by sequential cleavage of the amyloid precursor protein (APP). Alterations in the subcellular targeting of APP are thought to affect the degree of Aß production. Sorting receptors, such as SorLA, convey subcellular targeting of APP. Dysfunction of SorLA, and likely of the related receptors SorCS1 and SorCS3, cause AD. Nevertheless, disease progression could also provoke altered expression of the receptors. Here, we assessed if Aß plaque formation promotes altered expression of SorLA, SorCS1 and SorCS3. We analyzed transcript levels during aging and after amyloidosis in brain areas characterized by early amyloid plaque formation in an AD mouse model (APPPS1) and wild types. We observed stable expression levels during aging (1-12 months). After plaque formation, SorCS1 and SorLA expression were markedly reduced in the frontal cerebral cortex and to a minor extent in the hippocampus, whereas SorCS3 expression was solely reduced in the frontal cerebral cortex. Our results indicate that disease progression, associated with Aß accumulation, can negatively regulate expression of the receptors.

A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by phosphorylation sites.

Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.

Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, ß- and γ-secretases is a determining factor in Alzheimer's disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aß production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aß production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.

APLP1 Is a Synaptic Cell Adhesion Molecule, Supporting Maintenance of Dendritic Spines and Basal Synaptic Transmission.

The amyloid precursor protein (APP), a key player in Alzheimer's disease, belongs to the family of synaptic adhesion molecules (SAMs) due to its impact on synapse formation and synaptic plasticity. These functions are mediated by both the secreted APP ectodomain that acts as a neurotrophic factor and full-length APP forming trans-cellular dimers. Two homologs of APP exist in mammals: the APP like proteins APLP1 and APLP2, exhibiting functions that partly overlap with those of APP. Here we tested whether APLP1 and APLP2 also show features of SAMs. We found that all three family members were upregulated during postnatal development coinciding with synaptogenesis. We observed presynaptic and postsynaptic localization of all APP family members and could show that heterologous expression of APLP1 or APLP2 in non-neuronal cells induces presynaptic differentiation in contacting axons of cocultured neurons, similar to APP and other SAMs. Moreover, APP/APLPs all bind to synaptic-signaling molecules, such as MINT/X11. Furthermore, we report that aged APLP1 knock-out mice show impaired basal transmission and a reduced mEPSC frequency, likely resulting from reduced spine density. This demonstrates an essential nonredundant function of APLP1 at the synapse. Compared to APP, APLP1 exhibits increased trans-cellular binding and elevated cell-surface levels due to reduced endocytosis. In conclusion, our results establish that APLPs show typical features of SAMs and indicate that increased surface expression, as observed for APLP1, is essential for proper synapse formation in vitro and synapse maintenance in vivoSIGNIFICANCE STATEMENT According to the amyloid-cascade hypothesis, Alzheimer's disease is caused by the accumulation of Aß peptides derived from sequential cleavage of the amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Here we show that all mammalian APP family members (APP, APLP1, and APLP2) exhibit synaptogenic activity, involving trans-synaptic dimerization, similar to other synaptic cell adhesion molecules, such as Neuroligin/Neurexin. Importantly, our study revealed that the loss of APLP1, which is one of the major substrates of BACE1, causes reduced spine density in aged mice. Because some therapeutic interventions target APP processing (e.g., BACE inhibitors), those strategies may alter APP/APLP physiological function. This should be taken into account for the development of pharmaceutical treatments of Alzheimer's disease.

Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

SorCS1 variants and amyloid precursor protein (APP) are co-transported in neurons but only SorCS1c modulates anterograde APP transport.

Processing of amyloid precursor protein (APP) into amyloid-ß peptide (Aß) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aß is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface-bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co-immunoprecipitation and co-localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post-endocytic pathway. We introduce functional Venus-tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP-positive transport vesicles contain SorCS1. Co-expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP-positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles. Altered APP trafficking is thought to modulate its processing. SorCS1 has been suggested to function in APP trafficking. We analyzed if the two SorCS1 variants, SorCS1b and SorCS1c, tie APP to the cell surface or modify its internalization and intracellular targeting. We observed co-localization and vesicular co-transport of APP and SorCS1, but independent internalization and sorting through a common post-endocytic pathway. Co-expression of one variant, SorCS1c, reduced anterograde APP transport. These data demonstrate that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.

Differential role of APP and APLPs for neuromuscular synaptic morphology and function.

The analysis of mouse models indicated that APP and the related APLPs are important for synapse formation and function. The synaptic role of APP is, however, complex due to partially overlapping functions within the gene family. APP/APLPs are proteolytically cleaved and have both adhesive and signaling properties. Mice lacking individual APP family members are viable, whereas APP/APLP2 and APLP1/APLP2 double knockout (DKO) mice die shortly after birth. Here, we analyzed the morphology of the neuromuscular junction (NMJ) of lethal APLP1/APLP2-DKO mice in comparison to lethal APP/APLP2-DKO mutants and viable single KO mice. We report that, surprisingly, the NMJ phenotype of APLP1/APLP2-DKO mice shows striking differences as compared to APP/APLP2-DKO mice. Unexpectedly, APLP1/APLP2-DKO mice exhibit normal endplate patterning and lack presynaptic nerve terminal sprouting. However, at the level of individual synapses we show that APLP1/APLP2-DKO mice exhibit reduced size of pre- and postsynaptic compartments and reduced colocalization. As APP/APLP2-DKO and APLP1/APLP2-DKO mice show similar penetrance of early postnatal lethality, this suggests that deficits at the level of individual synapses due to impaired synaptic apposition and/or deficits in transmitter release may cause lethality. Using an in vitro cell-adhesion assay, we observed that APP trans-dimerization is considerably less efficient than APLP2 trans-interaction. Thus, differences between APP/APLP2 and APP/APLP1 NMJ formation may be in part explained by differences in APP/APLP2 trans-dimerization properties. Collectively, our study further highlights the distinct and essential role of APLP2 at NMJ synapses that cannot be compensated by APP.

A presenilin 1 mutation in the first case of Alzheimer's disease: revisited.

BACKGROUND: Recently, a single point mutation in the presenilin 1 (PSEN1) gene of the first described Alzheimer's disease (AD) patient Auguste D was reported by Müller and co-workers. However, the sequencing results of the DNA from a 100-year-old tissue contained some uncertainties. METHODS: We heat extracted DNA from an original histological slice of Auguste D's brain and used nested polymerase chain reaction for the amplification of different exons of genes known to be affected in familial forms of AD. RESULTS: Our sequencing analysis did not validate the reported mutation. Furthermore, an extended sequencing analysis of Auguste D's DNA revealed no indication of a nonsynonymous hetero- or homozygous mutation in the exons of APP, PSEN1, and PSEN2 genes comprising the already known familial AD mutations. CONCLUSION: Despite the wealth of data from Müller and co-workers, our results emphasize the requirement of more detailed analysis of Auguste D's DNA in future.