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Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ-secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full-length holoprotein forms, in particular familial Alzheimer's disease-causing forms of PS1, result in significantly attenuated calcium release from thapsigargin- and bradykinin-sensitive stores. Interestingly, treatment of HEK293 cells with γ-secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN-2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.

J. Neurochem. (2011) 119, 1064–1073. Abstract Mutations in presenilins are the major cause of familial Alzheimer’s disease (FAD), leading to impairments of memory and synaptic plasticity followed by age‐dependent neurodegeneration. Presenilins are the catalytic subunits of γ‐secretase, which itself is critically involved in the processing of amyloid precursor protein to release neurotoxic amyloid β (Aβ). Besides Aβ generation, there is growing evidence that presenilins play an essential role in the formation and maintenance of synapses. To further elucidate the effect of presenilin1 (PS1) on synapses, we performed longitudinal in vivo two‐photon imaging of dendritic spines in the somatosensory cortex of transgenic mice over‐expressing either human wild‐type PS1 or the FAD‐mutated variant A246E (FAD‐PS1). Interestingly, the consequences of transgene expression were different in two subtypes of cortical dendrites. On apical layer 5 dendrites, we found an enhanced spine density in both mice over‐expressing human wild‐type presenilin1 and FAD‐PS1, whereas on basal layer 3 dendrites only over‐expression of FAD‐PS1 increased the spine density. Time‐lapse imaging revealed no differences in kinetically distinct classes of dendritic spines nor was the shape of spines affected. Although γ‐secretase‐dependent processing of synapse‐relevant proteins seemed to be unaltered, higher expression levels of ryanodine receptors suggest a modified Ca2+ homeostasis in PS1 over‐expressing mice. However, the conditional depletion of PS1 in single cortical neurons had no observable impact on dendritic spines. In consequence, our results favor the view that PS1 influences dendritic spine plasticity in a gain‐of‐function but γ‐secretase‐independent manner.

Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.

Background: Alzheimer's disease (AD) is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP) plays a central role in AD. Owing to varying APP processing, several β-amyloid peptides (Aβ) are generated. In contrast to the form with 40 amino acids (Aβ 40 ), the variant with 42 amino acids (Aβ 42 ) is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Aβ effects have been studied extensively, little is known about specific genome-wide effects triggered by Aβ 42 or Aβ 40 derived from their direct precursor C99.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (Aβ) peptides by decreasing the cleavage of amyloid precursor protein (APP) by β-secretase, without notably affecting α- and γ-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates.

Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid β (Aβ) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics.