Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Genome-scale CRISPR screen reveals neddylation to contribute to cisplatin resistance of testicular germ cell tumours.

BACKGROUND: Type II testicular germ cell tumours (TGCT) are the most prevalent tumours in young men. Patients suffering from cisplatin-resistant TGCTs are facing very poor prognosis demanding novel therapeutic options. Neddylation is a known posttranslational modification mediating many important biological processes, including tumorigenesis. Overactivation of the neddylation pathway promotes carcinogenesis and tumour progression in various entities by inducing proteasomal degradation of tumour suppressors (e.g., p21, p27). METHODS: We used a genome-scale CRISPR/Cas9 activation screen to identify cisplatin resistance factors. TGCT cell lines were treated with the neddylation inhibitor (MLN4924)/cisplatin/combination and investigated for changes in viability (XTT assay), apoptosis/cell cycle (flow cytometry) as well as in the transcriptome (3'mRNA sequencing). RESULTS: NAE1 overexpression was detected in cisplatin-resistant colonies from the CRISPR screen. Inhibition of neddylation using MLN4924 increased cisplatin cytotoxicity in TGCT cell lines and sensitised cisplatin-resistant cells towards cisplatin. Apoptosis, G2/M-phase cell cycle arrest, γH2A.X/P27 accumulation and mesoderm/endoderm differentiation were observed in TGCT cells, while fibroblast cells were unaffected. CONCLUSIONS: We identified overactivation of neddylation as a factor for cisplatin resistance in TGCTs and highlighted the additive effect of NAE1 inhibition by MLN4924 in combination with cisplatin as a novel treatment option for TGCTs.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion.

The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.

Nanoparticles' properties modify cell type-dependent distribution in immune cells.

Polymeric nanoparticles can passively target inflamed tissues. How their physicochemical properties affect their distribution pattern among the infiltrating immune cells is unknown. Polyvinyl acetate nanoparticles with different particle size (100 and 300 nm) and surface charge (cationic, non-ionic, and anionic) were prepared and incubated with either LPS-activated or unactivated murine splenocytes. Nanoparticle association with macrophages, dendritic cells, neutrophils, B and T cells was investigated using flow cytometry. Cells associated with nanoparticles as follows: cationic>anionic>non-ionic and 300 nm > 100 nm. 40% of ionic nanoparticles were distributed among unactivated macrophages, reduced to 25% for activated macrophages. 60% of 100 nm and 40% of 300 nm non-ionic nanoparticles were distributed among unactivated and LPS-activated macrophages. This study highlights that particles' physicochemical properties impact the number of nanoparticles associating with immune cells more than their distribution pattern, which is principally determined by the cell activation state. This suggests a disease-dependent distribution pattern for therapeutic nanoparticles.

Modulation of Nanostructure-Based Lipopolysaccharide Active Immunotherapy in Cancer: Size and Composition Determine Short- and Long-Term Tolerability.

Despite holding promise for cancer immunotherapy, the strong pro-inflammatory properties of lipopolysaccharide (LPS) also account for severe localized and systemic side effects, restricting its administrable dosage and the possibility of chronic dosing. Herein, we exploited the surface-active properties of LPS molecules to develop pathogen-mimicking LPS-decorated nanostructures with different compositions (lipid nanoemulsion vs polymeric nanospheres) and sizes (volumetric mean diameters of 100 nm vs 700 nm). The formulations were tested in cell culture for their immunostimulatory properties and in vivo against a murine subcutaneous colorectal cancer model. While all nanostructures resulted in similar levels of apoptotic cell death in tumor cells cultured with splenocytes, both the size and the composition of the nanostructures were found to govern the short- and long-term tolerability of LPS-based immunotherapy in vivo. The toxicity-related end point of the animal trials was decided upon in the case of a body condition score (BCS) of 1 and poor hair coat, or more than 15% loss of the original body weight, while in the absence of long-term intolerability, the experiments were terminated in the case of full remission or once the tumor surpassed a volume of 1000 mm3. Size was an important determinant of short-term tolerability, with larger particles being associated with higher incidence and extent of localized necrosis (3-6% necrotic surface area). Nanostructure composition, on the other hand, predominantly governed the long-term systemic tolerability. Within this context, the higher affinity of LPS molecules to the triglyceride core of the nanoemulsion compared to the polymeric matrix significantly improved the tolerability of the former over time. In fact, the mean survival estimate of the animals treated with small LPS nanoemulsion (LPS-NE (small)) was at least 42 days longer than that of the LPS and the LPS-decorated polymeric nanoparticle (LPS-NP) groups. Unlike other treatment groups, the experiments on 80% of the animals in LPS-NE (small) were terminated due to complete remission or tumor volume >1000 mm3. While a better understanding of these findings requires a larger scale, mechanistic-oriented trial on larger animal models, they indicate the role of nanostructures as beyond the carriers of the incorporated immunotherapeutic cargos. This highlights the importance of a wise selection of nanoparticle composition and a purposeful tuning of their physicochemical properties to enhance the safety profile and improve the eventual immunotherapeutic outcome.

Cargo-free particles of ammonio methacrylate copolymers: From pharmaceutical inactive ingredients to effective anticancer immunotherapeutics.

Nanoparticles create exciting platforms for anticancer immunotherapy and vaccination, though their inherent immunomodulatory properties have remained underexploited. Ammonio methacrylate copolymers (AMC) are well-established excipients in pharmaceutical industry and components of controlled-release oral formulations. Here, we demonstrate that nanoscaling of type A and B AMC (Eudragit® RL and RS) endows these inactive ingredients immunostimulatory properties exploitable for cancer therapy. The particles induce the secretion of various pro-inflammatory cytokines and chemokines from the cells of innate immunity. Though the underlying mechanisms are not fully uncovered, the current work established the partial involvement of Toll-like Receptor 4 (TLR4) and Nuclear factor κB (NF-κB). The size and charge-dependency of the particles' pro-inflammatory properties and cytokine/chemokine induction profile was also demonstrated. Within the context of cancer immunotherapy, biweekly peritumoral nanoparticle injection led to a complete regression of the syngeneic colorectal tumor, or a significant growth retardation thereof, considerably extending the survival of tumor-bearing animals. Additionally, presence of the immunological memory in treated animals was established. Given their better economical and relatively safer profile compared to well-established chemo- and immunotheraputics, and their ability to serve as carriers for drug targeting, vaccination and combination therapy, AMC nanoparticles (AMCNP) are fascinating subjects for further research in the field of cancer therapy.

Nanoparticle-based delivery enhances anti-inflammatory effect of low molecular weight heparin in experimental ulcerative colitis.

Epithelial administration of low molecular weight heparin (LMWH) has proven its therapeutic efficiency in ulcerative colitis (UC) but still lacks of a sufficiently selective drug delivery system. Polymeric nanoparticles were used here not only to protect LMWH from intestinal degradation but also to provide targeted delivery to inflamed tissue in experimental colitis mice. LMWH was associated with polymethacrylate nanoparticles (NP) type A (PEMT-A) or type B (PEMT-B) of a size: 150 nm resulting in a maximum drug loading: 0.1 mg/mg. In a lipopolysaccharide-stimulated macrophages both, free LMWH and LMWH-NP have significantly reduced the cytokines secretion independently from cellular uptake. The in-vivo therapeutic efficiency was dose dependent as at low doses (100 IU/kg) only minor differences between free LMWH and LMWH-NP were found and the superiority of LMWH-NP became prominent with dose increase (500 IU/kg). Administration of LMWH-NP at 500 IU/kg has markedly improved the clinical activity as compared to LMWH while similarly pathophysiological indicators revealed increased therapeutic outcome in presence of NP compared to LMWH alone: Myeloperoxidase (Colitis control: 10 480 ± 5335, LMWH-PEMT-A NP: 1507 ± 2165, LMWH-PEMT-B NP: 382 ± 143, LMWH: 8549 ± 5021 units/g) and tumor necrosis factor: (Colitis control: 1636 ± 544, LMWH-PEMT-A NP: 511 ± 506, LMWH-PEMT-B NP: 435 ± 473, LMWH: 1110 ± 309 pg/g). Associating LMWH with NP is improving the anti-inflammatory efficiency of LMWH in-vivo by its protection against degradation in luminal environment and selective drug delivery. Such a combination holds promise for a highly specific therapy by its double selectivity towards the inflamed intestinal tissue. LMWH-PEMT NP have significantly improved the clinical activity in-vivo in comparison to free LMWH.

In vivo imaging demonstrates dendritic spine stabilization by SynCAM 1.

Formation and stability of synapses are required for proper brain function. While it is well established that synaptic adhesion molecules are important regulators of synapse formation, their specific role during different phases of synapse development remains unclear. To investigate the function of the synaptic cell adhesion molecule SynCAM 1 in the formation, stability, and maintenance of spines we used 2-photon in vivo imaging to follow individual spines over a long period of time. In SynCAM 1 knockout mice the survival rate of existing spines was reduced and fewer filopodia-like structures were converted into stable spines. SynCAM 1(flag) overexpression resulted in more stable spines and fewer filopodia-like structures. When SynCAM 1(flag) overexpression is turned on the spine density rapidly increases within a few days. Interestingly, the spine density stayed at an elevated level when SynCAM 1(flag) overexpression was turned off. Our data indicate that the SynCAM 1 induced altered spine density is not caused by the formation of newly emerging protrusions, instead SynCAM 1 stabilizes nascent synaptic contacts which promotes their maturation. Concomitant with the synaptic stabilization, SynCAM 1 generally prolongs the lifetime of spines. In summary, we demonstrate that SynCAM 1 is a key regulator of spine stability.

SynCAM 1 improves survival of adult-born neurons by accelerating synapse maturation.

The survival of adult-born dentate gyrus granule cells critically depends on their synaptic integration into the existing neuronal network. Excitatory inputs are thought to increase the survival rate of adult born neurons. Therefore, whether enhancing the stability of newly formed excitatory synapses by overexpressing the synaptic cell adhesion molecule SynCAM 1 improves the survival of adult-born neurons was tested. Here it is shown that overexpression of SynCAM 1 improves survival of adult-born neurons, but has no effect on the proliferation rate of precursor cells. As expected, overexpression of SynCAM 1 increased the synapse density in adult-born granule neurons. While adult-born granule neurons have very few functional synapses 15 days after birth, it was found that at this age adult-born neurons in SynCAM 1 overexpressing mice exhibited around three times more excitatory synapses, which were stronger than synapses of adult-born neurons of control littermates. In summary, the data indicated that additional SynCAM 1 accelerated synapse maturation, which improved the stability of newly formed synapses and in turn increased the likelihood of survival of adult-born neurons.

Topographic Mapping of the Synaptic Cleft into Adhesive Nanodomains.

The cleft is an integral part of synapses, yet its macromolecular organization remains unclear. We show here that the cleft of excitatory synapses exhibits a distinct density profile as measured by cryoelectron tomography (cryo-ET). Aiming for molecular insights, we analyzed the synapse-organizing proteins Synaptic Cell Adhesion Molecule 1 (SynCAM 1) and EphB2. Cryo-ET of SynCAM 1 knockout and overexpressor synapses showed that this immunoglobulin protein shapes the cleft's edge. SynCAM 1 delineates the postsynaptic perimeter as determined by immunoelectron microscopy and super-resolution imaging. In contrast, the EphB2 receptor tyrosine kinase is enriched deeper within the postsynaptic area. Unexpectedly, SynCAM 1 can form ensembles proximal to postsynaptic densities, and synapses containing these ensembles were larger. Postsynaptic SynCAM 1 surface puncta were not static but became enlarged after a long-term depression paradigm. These results support that the synaptic cleft is organized on a nanoscale into sub-compartments marked by distinct trans-synaptic complexes.

SynCAM 1 adhesion dynamically regulates synapse number and impacts plasticity and learning.

Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.

Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice.

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.

SynCAMs organize synapses through heterophilic adhesion.

Synapses are asymmetric cell junctions with precisely juxtaposed presynaptic and postsynaptic sides. Transsynaptic adhesion complexes are thought to organize developing synapses. The molecular composition of these complexes, however, remains incompletely understood, precluding us from understanding how adhesion across the synaptic cleft guides synapse development. Here, we define two immunoglobulin superfamily members, SynCAM 1 and 2, that are expressed in neurons in the developing brain and localize to excitatory and inhibitory synapses. They function as cell adhesion molecules and assemble with each other across the synaptic cleft into a specific, transsynaptic SynCAM 1/2 complex. Additionally, SynCAM 1 and 2 promote functional synapses as they increase the number of active presynaptic terminals and enhance excitatory neurotransmission. The interaction of SynCAM 1 and 2 is affected by glycosylation, indicating regulation of this adhesion complex by posttranslational modification. The SynCAM 1/2 complex is representative for the highly defined adhesive patterns of this protein family, the four members of which are expressed in neurons in divergent expression profiles. SynCAMs 1, 2, and 3 each can bind themselves, yet preferentially assemble into specific, heterophilic complexes as shown for the synaptic SynCAM 1/2 interaction and a second complex comprising SynCAM 3 and 4. Our results define SynCAM proteins as components of novel heterophilic transsynaptic adhesion complexes that set up asymmetric interactions, with SynCAM proteins contributing to synapse organization and function.

Molecular structure and physiological function of chloride channels.

Cl- channels reside both in the plasma membrane and in intracellular organelles. Their functions range from ion homeostasis to cell volume regulation, transepithelial transport, and regulation of electrical excitability. Their physiological roles are impressively illustrated by various inherited diseases and knock-out mouse models. Thus the loss of distinct Cl- channels leads to an impairment of transepithelial transport in cystic fibrosis and Bartter's syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and impaired endocytosis in Dent's disease, and to impaired extracellular acidification by osteoclasts and osteopetrosis. The disruption of several Cl- channels in mice results in blindness. Several classes of Cl- channels have not yet been identified at the molecular level. Three molecularly distinct Cl- channel families (CLC, CFTR, and ligand-gated GABA and glycine receptors) are well established. Mutagenesis and functional studies have yielded considerable insights into their structure and function. Recently, the detailed structure of bacterial CLC proteins was determined by X-ray analysis of three-dimensional crystals. Nonetheless, they are less well understood than cation channels and show remarkably different biophysical and structural properties. Other gene families (CLIC or CLCA) were also reported to encode Cl- channels but are less well characterized. This review focuses on molecularly identified Cl- channels and their physiological roles.