Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown. Here, we developed a microanalytical proteomics pipeline using capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) in the discovery setting to quantify developmental changes in the chief circadian pacemaker, the suprachiasmatic nucleus (SCN), before and after the onset of photoreceptor-dependent visual function. Nesting CE-ESI with trapped ion mobility spectrometry time-of-flight (TOF) mass spectrometry (TimsTOF PRO) doubled the number of identified and quantified proteins compared to the TOF-only control on the same analytical platform. From 10 ng of peptide input, corresponding to <∼0.5% of the total local tissue proteome, technical triplicate analyses identified 1894 proteins and quantified 1066 proteins, including many with important canonical functions in axon guidance, synapse function, glial cell maturation, and extracellular matrix refinement. Label-free quantification revealed differential regulation for 166 proteins over development, with enrichment of axon guidance-associated proteins prior to eye-opening and synapse-associated protein enrichment after eye-opening. Super-resolution imaging of select proteins using STochastic Optical Reconstruction Microscopy (STORM) corroborated the MS results and showed that increased presynaptic protein abundance pre/post eye-opening in the SCN reflects a developmental increase in synapse number, but not presynaptic size or extrasynaptic protein expression. This work marks the first development and systematic application of TimsTOF PRO for CE-ESI-based microproteomics and the first integration of microanalytical CE-ESI TimsTOF PRO with volumetric super-resolution STORM imaging to expand the repertoire of technologies supporting analytical neuroscience.

The synaptic basis of activity-dependent eye-specific competition.

Binocular vision requires proper developmental wiring of eye-specific inputs to the brain. In the thalamus, axons from the two eyes initially overlap in the dorsal lateral geniculate nucleus and undergo activity-dependent competition to segregate into target domains. Here, we combine eye-specific tract tracing with volumetric super-resolution imaging to measure the nanoscale molecular reorganization of developing retinogeniculate eye-specific synapses in the mouse brain. We show there are eye-specific differences in presynaptic vesicle pool size and vesicle association with the active zone at the earliest stages of retinogeniculate refinement but find no evidence of eye-specific differences in subsynaptic domain number, size, or transsynaptic alignment across development. Genetic disruption of spontaneous retinal activity decreases retinogeniculate synapse density, delays the emergence eye-specific differences in vesicle organization, and disrupts subsynaptic domain maturation. These results suggest that activity-dependent eye-specific presynaptic maturation underlies synaptic competition in the mammalian visual system.

Prediction to the prognosis of children with neuroblastoma by nomogram based on the first-diagnosed inflammatory markers.

BACKGROUND: Patients with high-risk neuroblastoma (NB) have a poor prognosis. The prognostic significance of inflammatory biomarker-based nomograms for children with NB has not been previously studied. METHODS: Part of patients diagnosed with NB in our center from January 2016 to March 2022 were included in the study. Inflammatory biomarkers were primary outcome measures, including C-reactive protein (CRP), ferritin, neutrophil to lymphocyte ratio (NLR), and lymphocyte to monocyte ratio (LMR), platelet to lymphocyte ratio (PLR) and systemic immune-inflammation index (SII). Univariate and multivariate survival analyses were performed to assess the prognostic value of these indicators for overall survival (OS) in NB children, showing the Kaplan-Meier survival curves and plotting the nomogram. C-index were used to detect predictability. RESULTS: 93 NB patients were retrospectively analyzed. CRP, ferritin, NLR, PLR, and SII were significantly associated with OS of NB patients, while LMR were found to be not predictive of OS for NB patients. The established nomogram is well-calibrated, and the C-index is 0.731. CONCLUSION: Survival analysis found part of inflammatory biomarkers related to the prognosis of NB. The nomogram could be used as a convenient predictive tool in clinical practice to evaluate the prognosis of NB children at first diagnosis.

Volumetric super-resolution imaging by serial ultrasectioning and stochastic optical reconstruction microscopy in mouse neural tissue.

Here, we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models. For complete details on the use and execution of this protocol, please refer to Sigal et al. (2015).