Abnormal calcium signaling is a central pathological component of Alzheimer’s disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-β and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-β and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

Primary resistance drastically limits the clinical success of immune checkpoint blockade (ICB) in melanoma. Resistance to ICB may also develop when tumours relapse after targeted therapy. To identify cancer cell-intrinsic mechanisms driving resistance to ICB, we generated single-cell RNA-sequencing (scRNA-seq) data from a prospective longitudinal cohort of patients on ICB therapy, including an early time point obtained after only one cycle of treatment. Comparing these data with murine scRNA-seq datasets, we established a comprehensive view of the cellular architecture of the treatment-naïve melanoma ecosystem, and defined 6 evolutionarily conserved melanoma transcriptional metaprograms (Melanocytic or MEL, Mesenchymal-like or MES, Neural Crest-like, Antigen Presentation, Stress (hypoxia response) and Stress (p53 response)). Spatial multi-omics revealed a non-random geographic distribution of cell states that is, at least partly, driven by the tumour microenvironment. The single-cell data allowed unambiguous discrimination between melanoma MES cells and cancer-associated fibroblasts both in silico and in situ, a long-standing challenge in the field. Importantly, two of the melanoma transcriptional metaprograms were associated with divergent clinical responses to ICB. While the Antigen Presentation cell population was more abundant in tumours from patients who exhibited a clinical response to ICB, MES cells were significantly enriched in early on-treatment biopsies from non-responders, and their presence significantly predicted lack of response. Critically, we identified TCF4 (E2-2) as a master regulator of the MES program and suppressor of both MEL and Antigen Presentation programs. Targeting TCF4 expression in MES cells either genetically or pharmacologically using a bromodomain inhibitor increased immunogenicity and sensitivity to targeted therapy. This study describes an increasingly complex melanoma transcriptional landscape and its rapid evolution under ICB. It also identifies a putative biomarker of early response to ICB and an epigenetic therapeutic strategy that increases both immunogenicity of ICB-refractory melanoma and their sensitivity to targeted therapy.

The family of B-cell lymphoma-2 (Bcl-2) proteins exerts key functions in cellular health. Bcl-2 primarily acts in mitochondria where it controls the initiation of apoptosis. However, during the last decades, it has become clear that this family of proteins is also involved in controlling intracellular Ca2+ signaling, a critical process for the function of most cell types, including neurons. Several anti- and pro-apoptotic Bcl-2 family members are expressed in neurons and impact neuronal function. Importantly, expression levels of neuronal Bcl-2 proteins are affected by age. In this review, we focus on the emerging roles of Bcl-2 proteins in neuronal cells. Specifically, we discuss how their dysregulation contributes to the onset, development, and progression of neurodegeneration in the context of Alzheimer's disease (AD). Aberrant Ca2+ signaling plays an important role in the pathogenesis of AD, and we propose that dysregulation of the Bcl-2-Ca2+ signaling axis may contribute to the progression of AD and that herein, Bcl-2 may constitute a potential therapeutic target for the treatment of AD.

The quote "bring it back, bring it back, don't take it away from me" from Queen's Love of my life describes the function of the sorting receptor RER1, a 23 kDa protein with four transmembrane domains (TMDs) that localizes to the intermediate compartment and the cis-Golgi. From there it returns escaped proteins that are not supposed to leave the endoplasmic reticulum (ER) back to it. Unique about RER1 is its ability to recognize its ligands through binding motifs in TMDs. Among its substrates are ER-resident proteins, as well as unassembled subunits of multimeric complexes that are retrieved back into the ER, this way guarding the full assembly of their respective complexes. The basic mechanisms for RER1-dependent retrieval have been already elucidated some years ago in yeast. More recently, several important cargoes of RER1 have been described in mammalian cells, and the in vivo role of RER1 is being unveiled by using mouse models. In this Review, we give an overview of the cell biology of RER1 in different models, discuss its controversial role in the brain and provide an outlook on future directions for RER1 research.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

Amyloid-β (Aβ) plaques are a prominent pathological hallmark of Alzheimer's disease (AD). They consist of aggregated Aβ peptides, which are generated through sequential proteolytic processing of the transmembrane protein amyloid precursor protein (APP) and several Aβ-associated factors. Efficient clearance of Aβ from the brain is thought to be important to prevent the development and progression of AD. The ubiquitin-proteasome system (UPS) is one of the major pathways for protein breakdown in cells and it has been suggested that impaired UPS-mediated removal of protein aggregates could play an important role in the pathogenesis of AD. To study the effects of an impaired UPS on Aβ pathology in vivo, transgenic APPSwe/PS1ΔE9 mice (APPPS1) were crossed with transgenic mice expressing mutant ubiquitin (UBB+1), a protein-based inhibitor of the UPS. Surprisingly, the APPPS1/UBB+1 crossbreed showed a remarkable decrease in Aβ plaque load during aging. Further analysis showed that UBB+1 expression transiently restored PS1-NTF expression and γ-secretase activity in APPPS1 mice. Concurrently, UBB+1 decreased levels of β-APP-CTF, which is a γ-secretase substrate. Although UBB+1 reduced Aβ pathology in APPPS1 mice, it did not improve the behavioral deficits in these animals.

Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.

Extracellular, high signal-to-noise ratio recordings from electrogenic cells require a tight coupling between the cellular membrane and the recording electrode. Self assembled monolayers (SAMs) of alkanethiols functionalized with peptides were used in combination with micro- and nano-structured features on the sensor surface. This combination of surface chemistry and topography triggers a phagocytosis-like engulfment and ensures tight coupling. In this paper we report the results concerning usage of different SAMs and the influence of the peptide concentration towards cell adhesion and outgrowth. Later on, the optimized peptide functionalized SAMs were applied on micro- and nano-structured sensor surfaces. As a result, phagocytosis-like events could be shown using focused ion beam SEM and confocal fluorescence imaging.

Efficient functional coupling of neuronal cells and electronic sensors could result in hybrid bidirectional communication between neurons and computers (L.J. Breckenridge et al., Advantages of using microfabricated extracellular electrodes for in vitro neuronal recordings, J. Neurosci Res. 42 (1995), pp. 266-276; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457-10462). Such systems could enable us to gain insight into the mechanisms of neuro-degenerative diseases like Parkinson's and Alzheimer's disease in vitro or could be used to improve the function and efficiency of devices used in vivo, for example in Deep Brain Stimulation devices that are already used in the treatment of Parkinson's disease. One of the major challenges for the development of reliable neuro-electronic systems is to perform extracellular recordings of action potentials with a high signal-to-noise ratio. The poor quality of these recordings is caused by the culture medium, which is present in the cleft between the cell membrane and the sensor surface (P. Fromherz, Neuroelectronic interfacing: semiconductor chips with ion channels, nerve cells and brain, in Nanoelectronics and Information Technology, R. Waser, ed., Wiley-VCH, Berlin, 2003, pp. 781-810; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457-10462). In this article, we describe a method allowing a reduction of the distance between the membrane and the surface by combining surface chemistry and topography. We have developed a specialised surface chemistry, based on small laminin-derived peptides, which applied onto the topographical structures, triggers their engulfment by the cell membrane in a phagocytosis-like event. In the phagocytotic pit, the distance between the cell membrane and the sensor surface is believed to be minimal. We describe the surface chemistry used for the controlled immobilisation of the small peptides on the surface of the needle-like structures that are manufactured on the surface of electronic devices. PC12 neuro-blastoma cells and genetically-modified HeLa cells have been used to investigate the interaction between the cell membrane and the peptide functionalised topographical structures. The membrane-surface interaction was examined by means of electron microscopy and fluorescence microscopy.

Approaching biological questions by means of quantitative analysis is of increasing interest. Methods of rapid analysis and diagnostics have been developed a lot in the past. One example is screening methods used by pharmaceutical companies. To date several devices are on the market for high content screening task on an imaging basis. Practically that means it acquires pictures of for example cells or cellular organelles and uses image analysis tools to analyze these sets of pictures. Normally multiwell plates are used for screening, to test several parameters in parallel. Until recently publications on the topic of high content screening are rather rare and lack significant methodological information. This is probably due to the fact that this field was dominated by pharmaceutical companies and therefore knowledge was classified. Only few examples peak out from suppliers highlighting the potential of the technique (for review on that topic see [1]). As currently the Life Science field and especially neuroscience is moving more in that direction it is time to catch up. Very recently descriptions of quantitative methods for analysis of in vitro neurite outgrowth have been published [2,3]. However mainly neuronal cell lines have been used and studies on primary neurons are scarce (for example [4]). Cell models are rather convenient but are not displaying all the features of real neurons. Apart from that we have encountered that the neurons react much more sensitive to treatments and drugs than do cell lines. Therefore we see it inevitable to work directly with primary neurons. Here we would like to describe strategies to use these cells in imaging based cellular screens. These include protocols for stained cell lines over living neurons derived from transgenic mice and how to overcome problems like feeder layers.

Efficient integration of neuronal cells and electronic devices could result in hybrid bi-directional communication systems that would enable us to interact at fundamental levels with biological structures and gain insight in the mechanisms governing their functions. Such systems require a very tight coupling between the neuronal cell membrane and the surface of an electronic chip. In this paper we report an approach where the combination of specialized surface chemistry and the manipulation of biological processes, like a phagocytosis-like process, might improve this coupling. As a model, we used coated micro- and nano-sized beads and induced phagocytosis-like events by adding them to cultured cells. The development of the surface chemistry and the results obtained with beads functionalized with a laminin derived peptide are presented.