Surface Trafficking of APP and BACE in Live Cells

Amyloid‐β (Aβ)‐peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β‐site APP‐cleaving enzyme (BACE). Using live‐cell imaging of APP and BACE labeled with pH‐sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α‐secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual‐color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.      

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Synapse clusters are preferentially formed by synapses with large recycling pool sizes

Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1-43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.     

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

Spastin is a microtubule severing ATPase that regulates intracellular and axonal transport of vesicles. Intracellular vesicle trafficking was analyzed in differentiated SH‐SY5Y‐neuroblastoma cells, transfected with spastin wild‐type and three spastin mutations (ΔN, K388R, S44L) to investigate spastin‐mediated effects on the velocity of vesicles, stained with LysoTracker Red®. The vesicle velocity varied considerably between mutations and detailed analysis revealed up to five distinct velocity classes. Microtubule severing by overexpressed wild‐type spastin caused reduced vesicle velocity. S44L and ΔN mutations, which were functionally impaired, showed similar velocities as control cells. K388R‐transfected cells exhibited an intermediate velocity profile. The results support the idea that spastin mutations not only alter axonal transport, but in addition regulate intracellular trafficking in the cell soma as well. J. Cell. Physiol. 226: 362–368, 2011. © 2010 Wiley‐Liss, Inc.     

Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

Determination of axonal transport velocities via image cross- and autocorrelation

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. O. Welzel and D. Boening contributed equally to this work.     

Identification of novel functional inhibitors of acid sphingomyelinase

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 μM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans.     

The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer?5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.