The impact of psychostimulants on central and peripheral neuro-immune regulation: a scoping review of cytokine profiles and their implications for addiction.

It is now well-accepted that psychostimulants act on glial cells causing neuroinflammation and adding to the neurotoxic effects of such substances. Neuroinflammation can be described as an inflammatory response, within the CNS, mediated through several cytokines, reactive oxygen species, chemokines and other inflammatory markers. These inflammatory players, in particular cytokines, play important roles. Several studies have demonstrated that psychostimulants impact on cytokine production and release, both centrally and at the peripheral level. Nevertheless, the available data is often contradictory. Because understanding how cytokines are modulated by psychoactive substances seems crucial to perspective successful therapeutic interventions, here, we conducted a scoping review of the available literature. We have focused on how different psychostimulants impact on the cytokine profile. Publications were grouped according to the substance addressed (methamphetamine, cocaine, methylphenidate, MDMA or other amphetamines), the type of exposure and period of evaluation (acute, short- or long-term exposure, withdrawal, and reinstatement). Studies were further divided in those addressing central cytokines, circulating (peripheral) levels, or both. Our analysis showed that the classical pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were those more investigated. The majority of studies have reported increased levels of these cytokines in the central nervous system after acute or repeated drug. However, studies investigating cytokine levels during withdrawal or reinstatement have shown higher variability in their findings. Although we have identified fewer studies addressing circulating cytokines in humans, the available data suggest that the results may be more robust in animal models than in patients with problematic drug use. As a major conclusion, an extensive use of arrays for relevant cytokines should be considered to better determine which cytokines, upon the classical ones, may be involved in the progression from episodic use to the development of addiction. A concerted effort is still necessary to address the link between peripheral and central immune players, including from a longitudinal perspective. Until there, the identification of new biomarkers and therapeutic targets to envision personalized immune-based therapeutics will continue to be unlikely.

Neuron-Microglia Contact-Dependent Mechanisms Attenuate Methamphetamine-Induced Microglia Reactivity and Enhance Neuronal Plasticity.

Exposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP3R2-Ca2+-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.

Carbon Monoxide Modulation of Microglia-Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role.

Microglia, the 'resident immunocompetent cells' of the central nervous system (CNS), are key players in innate immunity, synaptic refinement and homeostasis. Dysfunctional microglia contribute heavily to creating a toxic inflammatory milieu, a driving factor in the pathophysiology of several CNS disorders. Therefore, strategies to modulate the microglial function are required to tackle exacerbated tissue inflammation. Carbon monoxide (CO), an endogenous gaseous molecule produced by the degradation of haem, has anti-inflammatory, anti-apoptotic, and pro-homeostatic and cytoprotective roles, among others. ALF-826A, a novel molybdenum-based CO-releasing molecule, was used for the assessment of neuron-microglia remote communication. Primary cultures of rat microglia and neurons, or the BV-2 microglial and CAD neuronal murine cell lines, were used to study the microglia-neuron interaction. An approach based on microglial-derived conditioned media in neuronal culture was applied. Medium derived from CO-treated microglia provided indirect neuroprotection against inflammation by limiting the lipopolysaccharide (LPS)-induced expression of reactivity markers (CD11b), the production of reactive oxygen species (ROS) and the secretion of inflammatory factors (TNF-α, nitrites). This consequently prevented neuronal cell death and maintained neuronal morphology. In contrast, in the absence of inflammatory stimulus, conditioned media from CO-treated microglia improved neuronal morphological complexity, which is an indirect manner of assessing neuronal function. Likewise, the microglial medium also prevented neuronal cell death induced by pro-oxidant tert-Butyl hydroperoxide (t-BHP). ALF-826 treatment reinforced microglia secretion of Interleukin-10 (IL-10) and adenosine, mediators that may protect against t-BHP stress in this remote communication model. Chemical inhibition of the adenosine receptors A2A and A1 reverted the CO-derived neuroprotective effect, further highlighting a role for CO in regulating neuron-microglia communication via purinergic signalling. Our findings indicate that CO has a modulatory role on microglia-to-neuron communication, promoting neuroprotection in a non-cell autonomous manner. CO enhances the microglial release of neurotrophic factors and blocks exacerbated microglial inflammation. CO improvement of microglial neurotrophism under non-inflammatory conditions is here described for the first time.

Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetamine.

Methamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented Meth-elicited microglia reactivity and re-established risk assessment behavior as tested by elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.

Functional Dissection of Mitosis Using Immortalized Fibroblasts from the Indian Muntjac, a Placental Mammal with Only Three Chromosomes.

During cell division in eukaryotes a microtubule-based network undergoes drastic changes and remodeling to assemble a mitotic spindle competent to segregate chromosomes. Several model systems have been widely used to dissect the molecular and structural mechanisms behind mitotic spindle assembly and function. These include budding and fission yeasts, which are ideal for genetic and molecular approaches, but show limitations in high-resolution live-cell imaging, while being evolutionarily distant from humans. On the other hand, systems that were historically used for their exceptional properties for live-cell imaging of mitosis (e.g., newt lung cells and Haemanthus endosperm cells) lack the necessary genomic tools for molecular studies. In a CRISPR-Cas9 era, human cultured cells have conquered the privilege to be positioned among the most powerful genetically manipulatable systems, but their high chromosome number remains a significant bottleneck for the molecular dissection of mitosis in mammals. We believe that we can significantly broaden this scenario by establishing a unique placental mammal model system that combines the powerful genetic tools and low chromosome number of fission yeast and Drosophila melanogaster, with the exceptional cytological features of a rat kangaroo cell. This system is based on hTERT-immortalized fibroblasts from a female Indian muntjac, a placental mammal with the lowest known chromosome number (n = 3). Here we describe a series of methodologies established in our laboratory for the study of mitosis in Indian muntjac. These include standard techniques such as immunofluorescence, western blotting, and FISH, but also several state-of-the-art methodologies, including live-cell imaging, cell confinement, RNAi, super-resolution STED microscopy, and laser microsurgery.

TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity.

Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1ß. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.

A mouse model reproducing the pathophysiology of neonatal group B streptococcal infection.

Group B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.

Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical Rho GTPases express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation.