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Abstract Background Migraine affects a significant fraction of the world population, yet its etiology is not completely understood. In vitro results highlighted thalamocortical and intra-cortical glutamatergic synaptic gain-of-function associated with a monogenic form of migraine (familial-hemiplegic-migraine-type-1: FHM1). However, how these alterations reverberate on cortical activity remains unclear. As altered responsivity to visual stimuli and abnormal processing of visual sensory information are common hallmarks of migraine, herein we investigated the effects of FHM1-driven synaptic alterations in the visual cortex of awake mice. Methods We recorded extracellular field potentials from the primary visual cortex (V1) of head-fixed awake FHM1 knock-in (n = 12) and wild type (n = 12) mice in response to square-wave gratings with different visual contrasts. Additionally, we reproduced in silico the obtained experimental results with a novel spiking neurons network model of mouse V1, by implementing in the model both the synaptic alterations characterizing the FHM1 genetic mouse model adopted. Results FHM1 mice displayed similar amplitude but slower temporal evolution of visual evoked potentials. Visual contrast stimuli induced a lower increase of multi-unit activity in FHM1 mice, while the amount of information content about contrast level remained, however, similar to WT. Spectral analysis of the local field potentials revealed an increase in the β/low γ range of WT mice following the abrupt reversal of contrast gratings. Such frequency range transitioned to the high γ range in FHM1 mice. Despite this change in the encoding channel, these oscillations preserved the amount of information conveyed about visual contrast. The computational model showed how these network effects may arise from a combination of changes in thalamocortical and intra-cortical synaptic transmission, with the former inducing a lower cortical activity and the latter inducing the higher frequencies ɣ oscillations. Conclusions Contrast-driven ɣ modulation in V1 activity occurs at a much higher frequency in FHM1. This is likely to play a role in the altered processing of visual information. Computational studies suggest that this shift is specifically due to enhanced cortical excitatory transmission. Our network model can help to shed light on the relationship between cellular and network levels of migraine neural alterations. Graphical Abstract

Abstract 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).

As microtubule-organizing centers (MTOCs), centrosomes play a pivotal role in cell division, neurodevelopment and neuronal maturation. Among centrosomal proteins, centrin-2 (CETN2) also contributes to DNA repair mechanisms which are fundamental to prevent genomic instability during neural stem cell pool expansion. Nevertheless, the expression profile of CETN2 in human neural stem cells and their progeny is currently unknown. To address this question, we interrogated a platform of human neuroepithelial stem (NES) cells derived from post mortem developing brain or established from pluripotent cells and demonstrated that while CETN2 retains its centrosomal location in proliferating NES cells, its expression pattern changes upon differentiation. In particular, we found that CETN2 is selectively expressed in mature astrocytes with a broad cytoplasmic distribution. We then extended our findings on human autoptic nervous tissue samples. We investigated CETN2 distribution in diverse anatomical areas along the rostro-caudal neuraxis and pointed out a peculiar topography of CETN2-labeled astrocytes in humans which was not appreciable in murine tissues, where CETN2 was mostly confined to ependymal cells. As a prototypical condition with glial overproliferation, we also explored CETN2 expression in glioblastoma multiforme (GBM), reporting a focal concentration of CETN2 in neoplastic astrocytes. This study expands CETN2 localization beyond centrosomes and reveals a unique expression pattern that makes it eligible as a novel astrocytic molecular marker, thus opening new roads to glial biology and human neural conditions.

In recent years, the direct interaction between cancer cells and tumor microenvironment (TME) has emerged as a crucial regulator of tumor growth and a promising therapeutic target. The TME, including the surrounding peritumoral regions, is dynamically modified during tumor progression and in response to therapies. However, the mechanisms regulating the crosstalk between malignant and non-malignant cells are still poorly understood, especially in the case of glioma, an aggressive form of brain tumor. The presence of unique brain-resident cell types, namely neurons and glial cells, and an exceptionally immunosuppressive microenvironment pose additional important challenges to the development of effective treatments targeting the TME. In this review, we provide an overview on the direct and indirect interplay between glioma and neuronal and glial cells, introducing new players and mechanisms that still deserve further investigation. We will focus on the effects of neural activity and glial response in controlling glioma cell behavior and discuss the potential of exploiting these cellular interactions to develop new therapeutic approaches with the aim to preserve proper brain functionality.

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.

Recent studies have demonstrated an active role for neurons in glioma progression. Specifically, peritumoral neurons establish functional excitatory synapses with glioma cells, and optogenetic stimulation of cortical pyramidal neurons drives tumor progression. However, the specific role of different subsets of cortical neurons, such as GABAergic interneurons, remains unexplored. Here, we directly compared the effects of optogenetic stimulation of pyramidal cells vs. fast-spiking, GABAergic neurons. In mice inoculated with GL261 cells into the motor cortex, we show that optogenetic stimulation of pyramidal neurons enhances glioma cell proliferation. In contrast, optogenetic stimulation of fast-spiking, parvalbumin-positive interneurons reduces proliferation as measured by BrdU incorporation and Ki67 immunolabelling. Since both principal cells and fast-spiking interneurons are directly activated by sensory afferent input, we next placed tumors in the occipital cortex to test the impact of visual stimulation/deprivation. We report that total lack of visual input via dark rearing enhances the density of proliferating glioma cells, while daily visual stimulation by gratings of different spatial frequencies and contrast reduces tumor growth. The effects of sensory input are region-specific, as visual deprivation has no significant effect on tumor proliferation in mice with gliomas in the motor cortex. We also report that sensory stimulation combined with temozolomide administration delays the loss of visual responses in peritumoral neurons. Altogether, these data demonstrate complex effects of different neuronal subtypes in the control of glioma proliferation.

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood–brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration.