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Background Head and neck squamous cell carcinoma (HNSCC) is composed of a heterogeneous group of tumors arising trough environmental carcinogens or infection by human papillomavirus (HPV). Treatment interventions such as immunotherapy and targeted therapy have shown clinical benefit in HNSCC patients. Despite these encouraging results, resistance to treatment is still observed in the majority of patients. Additionally, clinical effectiveness of treatment options has also been shown to be associated with HPV status. Here we investigate the tumoral and peripheral landscape of HPV(-) vs. HPV(+) head and neck cancers to identify features able to expand treatment options for patients with Viral- and Carcinogen-Driven Head and Neck Cancer. Methods Biopsies and serum samples derived from 502 primary and metastatic HNSCC patients were leveraged for genomic, proteomic and immunochemistry evaluations. Tumor biopsies from HNSCC patients commercially obtained (n=143) or derived from patients enrolled in CP1108 trial (n=19, NCT01693562) were profiled by gene expression. Primary tumor biopsies (N=198) from HNSCC have been assessed by Whole Exome Sequence (WES). Expression of immune markers including CD8, NKp46 was evaluated by immunohistochemistry (IHC) on 186 and 214 tumors biopsies, respectively. The expression of 80 immune related soluble factors was evaluated in serum derived from n=285 patients of HNSCC enrolled in EAGLE (NCT02369874), a randomized, open-label, study assessing Durvalumab and Tremelimumab vs. Standard of Care (SoC). Statistical comparison between HPV(+) vs. HPV (-) samples were conducted using R software. Results Patients with HPV(-) vs. HPV(+) HNSCC were characterized by worse prognosis. Increased levels of immunosuppressive factors including VEGF (p=0.01), IL-8 (p=0.02), IL6 (p=0.07) and macrophages chemo attractive factor CCL4 (p=0.07) was observed in the serum of HPV(-) vs HPV(+) HNSCC patients. In the tumor microenvironment, higher mRNA expression of immune signatures associated with MDSC, Cancer Associated Fibroblast (CAF), and Metalloproteinase (MMP) was observed in HPV(-) vs. HPV (+) HNSCC patients. In contrast, HNSCC HPV(+) patients were characterized by increased mRNA expression of DC signatures and IFNg related genes (i.e. CXCL9). No differential infiltration of T and NK cells (CD8+ and NKp46+) were found in HPV (-) vs. HPV(+) patients. Enrichments of mutations in EGFR, and DNA repair genes (PMS1, POLK, ATM) was observed in HPV(+) patients. On the contrary, enrichments of mutations in TP53 was observed in HPV(-) patients. Conclusions Deep evaluation of tumoral and peripheral landscape of viral- versus carcinogen-driven HNSCC might help understanding differential outcome of treatments regimens in HPV(+) vs HPV (-) HNSCC thus leading to novel therapeutic interventions. Trial Registration NCT01693562,NCT02369874 Ethics Approval The study was approved by Astrazeneca. Consent Patients provided written consent to perform evalutions here described.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

GABA is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity, or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development, when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent, but independent of BDNF signalling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain and aid experimental design in translational brain research.

The polarization of developing neurons to form axons and dendrites is required for establishing neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT co-localize in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 via the proteasome, while loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

Matricellular proteins are secreted, nonstructural proteins that regulate the extracellular matrix (ECM) and interactions between cells through modulation of growth factor signaling, cell adhesion, migration, and proliferation. Despite being well described in the context of nonneuronal tissues, recent studies have revealed that these molecules may also play instrumental roles in central nervous system (CNS) development and diseases. In this minireview, we discuss the matricellular protein families SPARC (secreted protein acidic and rich in cysteine), Hevin/SC1 (SPARC-like 1), TN-C (Tenascin C), TSP (Thrombospondin), and CCN (CYR61/CTGF/NOV), which are secreted by astrocytes during development. These proteins exhibit a reduced expression in adult CNS but are upregulated in reactive astrocytes following injury or disease, where they are well placed to modulate the repair processes such as tissue remodeling, axon regeneration, glial scar formation, angiogenesis, and rewiring of neural circuitry. Conversely, their reexpression in reactive astrocytes may also lead to detrimental effects and promote the progression of neurodegenerative diseases.

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

Summary The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling. JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1–4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification, it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1 associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.

Background Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material. Electronic supplementary material The online version of this article (doi:10.1186/s12907-016-0039-3) contains supplementary material, which is available to authorized users.