Quantitative Microproteomics Based Characterization of the Central and Peripheral Nervous System of a Mouse Model of Krabbe Disease

et al. 2020

Front. Cell. Neurosci.

Self Cite

Neuronal hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission, but the synaptic alterations leading to enhanced seizure propensity are only partly understood. Taking advantage of a mouse model of neocortical epilepsy, we used a combination of photoconversion and electron microscopy to assess changes in synaptic vesicles pools in vivo. Our analyses reveal that epileptic networks show an early onset lengthening of active zones at inhibitory synapses, together with a delayed spatial reorganization of recycled vesicles at excitatory synapses. Proteomics of synaptic content indicate that specific proteins were increased in epileptic mice. Altogether, our data reveal a complex landscape of nanoscale changes affecting the epileptic synaptic release machinery. In particular, our findings show that an altered positioning of release-competent vesicles represent a novel signature of epileptic networks.

Section: Methodsmentioning

confidence: 99%

Synaptic Vesicles Dynamics in Neocortical Epilepsy

Vannini

Restani

et al. 2020

Front. Cell. Neurosci.

Self Cite

“…Protein digestion was performed using a modified single-pot, solidphase-enhanced (SP3) sample preparation protocol [54][55][56] . A detailed description of the protocol is available in the Supplementary Information (Supplementary Methods S3).…”

Section: Proteomics Sample Preparationmentioning

confidence: 99%

Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model

Anastasi

Greco

et al. 2020

Sci Rep

Self Cite

Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1).

“…TMT sets underwent high pH fractionation on an AssayMap Bravo (Agilent technologies) and fractions run on a nano-LC (Easy1000 Thermo Fisher Scientific) equipped with a 50 cm EasySpray column and coupled with an Orbitrap Fusion for MS3 analysis (Thermo Fisher Scientific). Experimental details regarding sample fractionation and LC-MS/MS runs have been already reported elsewere (Pellegrini et al, 2019). Data were analysed using Proteome Discoverer 2.1.…”

Section: Proteomics Sample Preparation and Data Analysismentioning

confidence: 99%

Synaptic vesicles dynamics in neocortical epilepsy

Vannini

Restani

et al. 2020

Preprint

Self Cite

Neuronal networks’ hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission; however, underlying synaptic alterations leading to this condition remains poorly understood. Here, we assess synaptic changes in the visual cortex of epileptic tetanus neurotoxin-injected mice. Using an ultrastructural measure of synaptic activity, we quantified functional differences at excitatory and inhibitory synapses. We found homeostatic changes in hyperexcitable networks, expressed as an early onset lengthening of active zones at inhibitory synapses followed by spatial reorganization of recycled vesicles at excitatory synapses. A proteomic analysis of synaptic content revealed an upregulation of Carboxypeptidase E (CPE) following Tetanus NeuroToxin (TeNT) injection. Remarkably, inhibition of CPE rapidly decreased network discharges in vivo. These analyses reveal a complex landscape of homeostatic changes affecting the epileptic synaptic release machinery, differentially at inhibitory and excitatory terminals. Our study unveil homeostatic presynaptic mechanisms which may impact release timing rather than synaptic strength.