Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Electrical stimulation and recording from non-cranial areas in freely moving rodents often require tethered devices for monitoring from a distant location. Inspired by our experience in electrode implantation for deep brain stimulation (DBS), we developed an implantation approach, which allows for performing electrical modulation of the urinary bladder. In order to prevent the wires from being damaged by rats in freely moving conditions and compromising animal welfare, we guided the wires subcutaneously toward the nape to reach a plug, which was mounted on the skull. No complications such as infection, gastrointestinal tract injury, or inflammation occurred during or after surgery. The device allowed easy plugging and unplugging throughout the bladder stimulation and behavioral testing sessions undertaken in freely moving animals. This method enables modulation of peripheral organs in combination with DBS in freely moving rodents and can encompass many procedures such as electrical stimulation and recording, drug delivery, or electromyography.

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

Spinal cord injury (SCI) represents a major medical challenge. At present, there is still no cure to treat it efficiently and enable functional recovery below the injury site. Previously, we demonstrated that inflammation determines the fate of the physiopathology. To decipher the molecular mechanisms involved in this process, we performed a meta-analysis of our spatio-temporal proteomic studies in the time course of SCI. This highlighted the presence of IgG isotypes in both spinal cord explants and their secretomes. These IgGs were detected in the spinal cord even if no SCI occurred. However, during the time course following SCI, abundance of IgG1 and IgG2 subclasses (a, b, c) varied according to the spatial repartition. IgG1 was clearly mostly abundant at 12 h, and a switch to IgG2a was observed after 24 h. This IgG stayed predominant 3, 7, and 10 days after SCI. A protein related to IgM as well as a variable heavy chain were only detected 12 h after lesion. Interestingly, treatment with RhoA inhibitor influenced the abundance of the various IgG isotypes and a preferential switch to IgG2c was observed. By data reuse of rat dorsal root ganglion (DRG) neurons RNAseq datasets and RT-PCR experiments performed on cDNA from DRG sensory neurons ND7/23 and N27 dopaminergic neural cell lines, we confirmed expression of immunoglobulin heavy and light chains (constant and variable) encoding genes in neurons. We then identified CD16 and CD32b as their specific receptors in sensory neuron cell line ND7/23 and their activation regulated neurites outgrowth. These results suggest that during SCI, neuronal IgG isotypes are released to modulate neurites outgrowth. Therefore, we propose a new view of the SCI response involving an antibody dependent neurite outgrowth modulation (ADNM) which could be a precursor to the neuroinflammatory response in pathological conditions.

The skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. Significance: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness...), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization.

The skin as the interface with the external environment plays a crucial role in host defense against microbial attack. Using this unique feature, skin has become a promising site for vaccine administration. When adaptive immunity is well known, early innate molecular events are poorly understood. We aim to determine novel biomarkers of skin reaction following intradermal (i.d.) immunization. Ex vivo human explant model from healthy donors was used to investigate by NanoLC-MS/MS and MALDI imaging analysis, the early innate molecular events produced at the i.d. Influenza trivalent vaccine (TIV) inoculation site. In this proof-of-concept study, we identified early innate immune proteins differentially expressed in dermal and epidermal layer at 4 to 18 hours following immunization compared to baseline. Up-regulated top network proteins in the dermis were related to cell-to-cell signaling and cell trafficking, whereas in the epidermis, they were associated with antigen-presenting cells at early time point following immunization. In the dermis, CXCL8 chemokines signature was detected following dermal immunization. In the epidermis, CXCL10 linked network proteins was confirmed by qPCR as markers of antigen-presenting cell activation. In addition, skin MALDI imaging allowed to localize in the tissue major metabolites related to these signatures. Our study proposes novel step-forward approach for the identification of biomarkers during early innate reaction at the site of immunization that could correlate with immune protection

See Cunha (doi:10.1093/brain/awz335) for a scientific commentary on this article. Carvalho et al. provide clues to the onset of immune dysregulation underlying early synaptic loss in Alzheimer’s disease and tauopathies, by linking overactivation of adenosine A2A receptors in tau pathology to a particular microglial signature (upregulation of C1q and TREM2) allied to the loss of glutamatergic synapses and cognitive deficits.

Reflexes, that involve the spinobulbospinal pathway control both storage and voiding of urine. The periaqueductal gray matter (PAG), a pontine structure is part of the micturition pathway. Alteration in this pathway could lead to micturition disorders and urinary incontinence, such as the overactive bladder symptom complex (OABS). Although different therapeutic options exist for the management of OABS, these are either not effective in all patients. Part of the pathology of OABS is faulty sensory signaling about the filling status of the urinary bladder, which results in aberrant efferent signaling leading to overt detrusor contractions and the sensation of urgency and frequent voiding. In order to identify novel targets for therapy (i.e., structures in the central nervous system) and explore novel treatment modalities such as neuromodulation, we aimed at investigating which areas in the central nervous system are functionally activated upon sensory afferent stimulation of the bladder. Hence, we designed a robust protocol with multiple readout parameters including immunohistological and behavioral parameters during electrical stimulation of the rat urinary bladder. Bladder stimulation induced by electrical stimulation, below the voiding threshold, influences neural activity in: (1) the caudal ventrolateral PAG, close to the aqueduct; (2) the pontine micturition center and locus coeruleus; and (3) the superficial layers of the dorsal horn, sacral parasympathetic nucleus and central canal region of the spinal cord. In stimulated animals, a higher voiding frequency was observed but was not accompanied by increase in anxiety level and locomotor deficits. Taken together, this work establishes a critical role for the vlPAG in the processing of sensory information from the urinary bladder and urges future studies to investigate the potential of neuromodulatory approaches for urological diseases.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in elderly people. AD is characterized by a progressive cognitive decline and it is neuropathologically defined by two hallmarks: extracellular deposits of aggregated β-amyloid (Aβ) peptides and intraneuronal fibrillar aggregates of hyper- and abnormally phosphorylated Tau proteins. AD results from multiple genetic and environmental risk factors. Epidemiological studies reported beneficial effects of caffeine, a non-selective adenosine receptors antagonist. In the present review, we discuss the impact of caffeine and of adenosinergic system modulation on AD, in terms of pathology and therapeutics.

OBJECTIVES:To determine the phenotype of the ventrolateral part of the periaqueductal gray matter neurons after bladder stimulation.METHODS:In the experimental group, electrical stimulation of the bladder was carried out under freely moving condition by a bipolar stimulation electrode implanted in the bladder wall. Thereafter, the brain sections were processed for immunohistochemical analysis using antibodies against c-Fos (neuronal activation marker) together with one of the following: tyrosine hydroxylase (dopaminergic cell marker), vesicular glutamate transporter (glutamatergic cell marker), serotonin, glutamate decarboxylase (glutamate decarboxylase 67, gamma-aminobutyric acid cell marker) and neuronal nitric oxide synthase. We used design-based confocal stereological analysis to quantify the immunohistochemically stained sections.RESULTS:A significant increase in the number of c-Fos-positive cells in the ventrolateral part of the periaqueductal gray matter after stimulation was found. Furthermore, the ratio of c-Fos cells double labeled with vesicular glutamate transporter was significantly higher in the ventrolateral part of the periaqueductal gray matter region in the stimulated compared with the sham group. Quantitative analysis of the other four cell types did not show any significant difference.CONCLUSION:These findings suggest that glutamatergic neurotransmission in the ventrolateral part of the periaqueductal gray matter is seemingly the main pathway to be activated after receiving sensory signals from the bladder.

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.

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

Objective: Mechanical and physical stimuli can evoke release of several substances by the urothelium, for example, prostaglandin E2 (PGE2), nitric oxide, acetylcholine and adenosine triphosphate. It has been shown, that PGE2 and acetylcholine seem to act in a positive feedback on molecular level. This feedback was located in the urothelial/suburothelial layer. Therefore, the aim of our study was to investigate the role of PGE2 and one of its receptors, EP1, on muscarinic induced contractions.Methods: the urethra and bladder of 24 male guinea pigs were dissected after sacrifice of the animal. The bladder was catheterised transurethrally and placed into a preheated organ bath filled with Krebs buffer. The bladders were repeatedly stimulated with the muscarinic agonist arecaidine alone or in combination with a) acetylsalicylic acid, b) PGE2 or c) the EP1 antagonist ONO-8713. All drugs were applied at the serosal side of the bladder. Intravesical pressure was measured and amplitude and frequency of the arecaidine induced response were analysed.Results: Adding PGE2 before muscarinic stimulations induced an amplifying effect on muscarinically induced contractions and inhibition of the PGE2 producing enzymes prior to an arecaidine stimulation decreased the amplitude during the initial phase of the response. Inhibition of EP1 resulted in a decreased frequency during the second phase of the arecaidine response.Conclusions: In summary, this study provides evidence that the cholinergic and prostanoid systems in the urinary bladder act in a positive feedback loop and that the EP1 receptor plays a subtle role in this process.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.