A distinct pattern of growth and RAC1 signaling in melanoma brain metastasis cells.

BACKGROUND: Melanoma, the deadliest of skin cancers, has a high propensity to form brain metastases that are associated with a markedly worsened prognosis. In spite of recent therapeutic advances, melanoma brain lesions remain a clinical challenge, biomarkers predicting brain dissemination are not clear and differences with other metastatic sites are poorly understood. METHODS: We examined a genetically diverse panel of human-derived melanoma brain metastasis (MBM) and extracranial cell lines using targeted sequencing, a Reverse Phase Protein Array, protein expression analyses, and functional studies in vitro and in vivo. RESULTS: Brain-specific genetic alterations were not detected; however, MBM cells in vitro displayed lower proliferation rates and MBM-specific protein expression patterns associated with proliferation, DNA damage, adhesion, and migration. MBM lines displayed higher levels of RAC1 expression, involving a distinct RAC1-PAK1-JNK1 signaling network. RAC1 knockdown or treatment with small molecule inhibitors contributed to a less aggressive MBM phenotype in vitro, while RAC1 knockdown in vivo led to reduced tumor volumes and delayed tumor appearance. Proliferation, adhesion, and migration were higher in MBM vs nonMBM lines in the presence of insulin or brain-derived factors and were affected by RAC1 levels. CONCLUSIONS: Our findings indicate that despite their genetic variability, MBM engage specific molecular processes such as RAC1 signaling to adapt to the brain microenvironment and this can be used for the molecular characterization and treatment of brain metastases.

A microRNA signature that correlates with cognition and is a target against cognitive decline.

While some individuals age without pathological memory impairments, others develop age-associated cognitive diseases. Since changes in cognitive function develop slowly over time in these patients, they are often diagnosed at an advanced stage of molecular pathology, a time point when causative treatments fail. Thus, there is great need for the identification of inexpensive and minimal invasive approaches that could be used for screening with the aim to identify individuals at risk for cognitive decline that can then undergo further diagnostics and eventually stratified therapies. In this study, we use an integrative approach combining the analysis of human data and mechanistic studies in model systems to identify a circulating 3-microRNA signature that reflects key processes linked to neural homeostasis and inform about cognitive status. We furthermore provide evidence that expression changes in this signature represent multiple mechanisms deregulated in the aging and diseased brain and are a suitable target for RNA therapeutics.

Differences in synaptic vesicle pool behavior between male and female hippocampal cultured neurons.

A strong focus on sex-related differences has arisen recently in neurobiology, but most investigations focus on brain function in vivo, ignoring common experimental models like cultured neurons. A few studies have addressed morphological differences between male and female neurons in culture, but very few works focused on functional aspects, and especially on presynaptic function. To fill this gap, we studied here functional parameters of synaptic vesicle recycling in hippocampal cultures from male and female rats, which are a standard model system for many laboratories. We found that, although the total vesicle pools are similar, the recycling pool of male synapses was larger, and was more frequently used. This was in line with the observation that the male synapses engaged in stronger local translation. Nevertheless, the general network activity of the neurons was similar, and only small differences could be found when stimulating the cultures. We also found only limited differences in several other assays. We conclude that, albeit these cultures are similar in behavior, future studies of synapse behavior in culture should take the sex of the animals into account.

The mRNA-Binding Protein RBM3 Regulates Activity Patterns and Local Synaptic Translation in Cultured Hippocampal Neurons.

The activity and the metabolism of the brain change rhythmically during the day/night cycle. Such rhythmicity is also observed in cultured neurons from the suprachiasmatic nucleus, which is a critical center in rhythm maintenance. However, this issue has not been extensively studied in cultures from areas less involved in timekeeping, as the hippocampus. Using neurons cultured from the hippocampi of newborn rats (both male and female), we observed significant time-dependent changes in global activity, in synaptic vesicle dynamics, in synapse size, and in synaptic mRNA amounts. A transcriptome analysis of the neurons, performed at different times over 24 h, revealed significant changes only for RNA-binding motif 3 (Rbm3). RBM3 amounts changed, especially in synapses. RBM3 knockdown altered synaptic vesicle dynamics and changed the neuronal activity patterns. This procedure also altered local translation in synapses, albeit it left the global cellular translation unaffected. We conclude that hippocampal cultured neurons can exhibit strong changes in their activity levels over 24 h, in an RBM3-dependent fashion.SIGNIFICANCE STATEMENT This work is important in several ways. First, the discovery of relatively regular activity patterns in hippocampal cultures implies that future studies using this common model will need to take the time parameter into account, to avoid misinterpretation. Second, our work links these changes in activity strongly to RBM3, in a fashion that is independent of the canonical clock mechanisms, which is a very surprising observation. Third, we describe here probably the first molecule (RBM3) whose manipulation affects translation specifically in synapses, and not at the whole-cell level. This is a key finding for the rapidly growing field of local synaptic translation.

Ultrastructural Correlates of Presynaptic Functional Heterogeneity in Hippocampal Synapses.

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.

A genetically specified connectomics approach applied to long-range feeding regulatory circuits.

Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase-expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.

De novo insertions and deletions of predominantly paternal origin are associated with autism spectrum disorder.

Whole-exome sequencing (WES) studies have demonstrated the contribution of de novo loss-of-function single-nucleotide variants (SNVs) to autism spectrum disorder (ASD). However, challenges in the reliable detection of de novo insertions and deletions (indels) have limited inclusion of these variants in prior analyses. By applying a robust indel detection method to WES data from 787 ASD families (2,963 individuals), we demonstrate that de novo frameshift indels contribute to ASD risk (OR = 1.6; 95% CI = 1.0-2.7; p = 0.03), are more common in female probands (p = 0.02), are enriched among genes encoding FMRP targets (p = 6 × 10(-9)), and arise predominantly on the paternal chromosome (p < 0.001). On the basis of mutation rates in probands versus unaffected siblings, we conclude that de novo frameshift indels contribute to risk in approximately 3% of individuals with ASD. Finally, by observing clustering of mutations in unrelated probands, we uncover two ASD-associated genes: KMT2E (MLL5), a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release.