In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV‐specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co‐isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV‐specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Tissue spatially-resolved proteomics was performed on 3 brain regions, leading to the characterization of 123 reference proteins. Moreover, 8 alternative proteins from alternative open reading frames (AltORF) were identified. Some proteins display specific post-translational modification profiles or truncation linked to the brain regions and their functions. Systems biology analysis performed on the proteome identified in each region allowed to associate sub-networks with the functional physiology of each brain region. Back correlation of the proteins identified by spatially-resolved proteomics at a given tissue localization with the MALDI MS imaging data, was then performed. As an example, mapping of the distribution of the matrix metallopeptidase 3-cleaved C-terminal fragment of α-synuclein (aa 95–140) identified its specific distribution along the hippocampal dentate gyrus. Taken together, we established the molecular physiome of 3 rat brain regions through reference and hidden proteome characterization.

Traumatic brain injury (TBI) represents a major health concerns with no clinically-approved FDA drug available for therapeutic intervention. Several genomics and neuroproteomics studies have been employed to decipher the underlying pathological mechanisms involved that can serve as potential neurotherapeutic targets and unveil a possible underlying relation of TBI to other secondary neurological disorders. In this work, we present a novel high throughput systems biology approach using a spatially resolved microproteomics platform conducted on different brain regions in an experimental rat model of moderate of controlled cortical injury (CCI) at a temporal pattern postinjury (1 day, 3 days, 7 days, and 10 days). Mapping the spatiotemporal landscape of signature markers in TBI revealed an overexpression of major protein families known to be implicated in Parkinson9s disease (PD) such as GPR158, HGMB1, synaptotagmin and glutamate decarboxylase in the ipsilateral substantia nigra. In silico bioinformatics docking experiments indicated the potential correlation between TBI and PD through alpha-synuclein. In an in vitro model, stimulation with palmitoylcarnitine triggered an inflammatory response in macrophages and a regeneration processes in astrocytes which also further confirmed the in vivo TBI proteomics data. Taken together, this is the first study to assess the microproteomics landscape in TBI, mainly in the substantia nigra, thus revealing a potential predisposition for PD or Parkinsonism post-TBI. Graphical Abstract Highlights Spatiotemporal microproteomics analysis of TBI. Injury site microproteomics reveal distinct phases in 10-day frame post TBI. Uninjured proteomic profile is restored in TBI at 10 days post injury. Substantia nigra protein post 3 days suggest link to Parkinson9s disease.

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-β) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.