Possible modulation of dopaminergic neurotransmission function by acetyl-L-carnitine

Acetyl‐L‐Carnitine (ALC) has a putative neuroprotective effect being used in a variety of conditions. Nevertheless, the underlying molecular mechanisms, particularly regarding the induction of changes in neurotransmitter systems, are still not fully understood. We aim to contribute for the elucidation of the mechanisms by which ALC alters neurotransmitter release, using a cell line and an animal model of exposure to methamphetamine (METH). PC12 cells were incubated with several doses of ALC (0.01 to 1.0 mM) alone or in combination with METH 1.0 or 100 µM for 24h or 72h. When combined, ALC preceded METH administration in 30 minutes. Dopamine (DA) content was determined by high performance liquid chromatography. C57BL/6J mice were used for in vivo assays to assess DA striatal binding. Mice were divided into 4 groups, according to different treatments: group 1 (control), group 2 (ALC, 100 mg/kg), group 3 (METH, 10 mg/kg) and group 4 (ALC+METH). Images were acquired in a SPECT/CT scanner (NanoSPECT/CT, Mediso, Hungary) 70 minutes after 123I‐IBZM injection. Regions of interest were drawn in the striata and in the cerebellum to determine the striatal binding ratio. Increased intracellular levels of DA were observed in PC12 cells at 24h and 72h after the administration of ALC. Cells treated with METH 100 µM displayed decreased intracellular levels of DA. ALC prevented the METH‐induced decrease in DA concentration (p<0.0001). On the other hand, a single dose of 10 mg/kg of METH induced a decrease in striatal D2R binding ratios comparing to control group (between 20% and 30%). Interestingly, over time, ALC was able to reverse the decrease on the radiotracer binding induced by METH. The present study indicates a possible effect of ALC over METH‐induced DA release.info:eu-repo/semantics/publishedVersio

Daily alcohol intake triggers aberrant synaptic pruning leading to synapse loss and anxiety-like behavior

Alcohol abuse adversely affects the lives of millions of people worldwide. Deficits in synaptic transmission and in microglial function are commonly found in human alcohol abusers and in animal models of alcohol intoxication. Here, we found that a protocol simulating chronic binge drinking in male mice resulted in aberrant synaptic pruning and substantial loss of excitatory synapses in the prefrontal cortex, which resulted in increased anxiety-like behavior. Mechanistically, alcohol intake increased the engulfment capacity of microglia in a manner dependent on the kinase Src, the subsequent activation of the transcription factor NF-κB, and the consequent production of the proinflammatory cytokine TNF. Pharmacological blockade of Src activation or of TNF production in microglia, genetic ablation of Tnf, or conditional ablation of microglia attenuated aberrant synaptic pruning, thereby preventing the neuronal and behavioral effects of the alcohol. Our data suggest that aberrant pruning of excitatory synapses by microglia may disrupt synaptic transmission in response to alcohol abuse.This work was financed by FEDER -Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 -Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-030647 (PTDC/SAU-TOX/30647/2017) in TS lab. The projects FEDER Portugal (Norte-01-0145-FEDER-000008000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); FCOMP-01-0124-FEDER-021333) and FCT (PTDC/MED-NEU/31318/2017) supported work in JBR lab. CCP and RS hold employment contracts financed by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., in the context of the program-contract described in paragraphs 4, 5 and 6 of art. 23 of Law no. 57/2016, of August 29, as amended by Law no. 57/2017 of July 2019. TC is supported by FCT (SFRH/BD/117148/2016). RLA was supported by FCT (PD/BD/114266/2016). AM was supported by FCT (IF/00753/2014). Author contributions: RS, TS, and JBR designed the project. RS, JFH, CCP, TOA, JTM, RLA, TC, CS, and AM performed experiments. RS, TS, and JBR co-supervised the study. RS and JBR wrote the original draft. RS, CCP, TS, and JBR reviewed and edited the manuscript. TS and JBR acquired funding

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.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT— Fundação para a Ciência e a Tecnologia/Ministério da Ciência (FCT), Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-030647 (PTDC/ SAU-TOX/30647/2017) in TS lab. FEDER Portugal (Norte-01-0145-FEDER000008000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); FCOMP-01-0124-FEDER-021333). CCP and RS hold employment contracts financed by national funds through FCT –in the context of the program-contract described in paragraphs 4, 5, and 6 of art. 23 of Law no. 57/ 2016, of August 29, as amended by Law no. 57/2017 of July 2019. TC, TOA, AFT, JB, AIS and AM were supported by FCT (SFRH/BD/117148/2016, SFRH/BD/147981/2019, 2020.07188.BD, PD/BD/135450/2017, SFRH/BD/144324/2019, and IF/00753/2014). Work in JBR lab was supported by the FCT project PTDC/ MED-NEU/31318/2017. JFO was also supported by FCT projects PTDC/MED-NEU/31417/2017 and POCI-01- 0145-FEDER-016818; Bial Foundation Grants 207/14 and 037/18, by National funds, through FCT - project UIDB/50026/2020; and by the projects NORTE-01-0145-FEDER000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Funding of i3S Scientific Platforms: Advanced Light Microscopy (ALM), a member of the national infrastructure PPBI-Portuguese Platform of BioImaging (POCI-01–0145-FEDER022122); and Genomics through GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT

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.The authors gratefully acknowledge the help of Encarnacao Ribeiro for excellent technical assistance, Joana Tavares for assisting with IVIS Lumina LT, Susana Roque for the luminex instrument experiments, the Molecular Microbiology group at i3S for microscope use, and the Portuguese architect and artist Gil Ferreira da Silva for the artworks included in the last figure. This work was supported by funds from Foundation for Science and Technology (FCT), European Regional Development Fund (FEDER) and Compete under project POCI-01-0145-FEDER-016607 (PTDC/IMI-MIC/1049/2014) and from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). T.S. and A.M. were supported by Investigador FCT (IF/00875/2012 and IF/00753/2014), POPH and Fundo Social Europeu. E.B.A. and C.C.P. hold postdoctoral fellowships from FCT (PTDC/IMI-MIC/1049/2014 and SFRH/BPD/91962/2012). Ar.F. and P.T.C. were supported by Laboratoire d'Excellence (LABEX) Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID)

Biology-oriented development of novel lipophilic antioxidants with neuroprotective activity

Hydroxycinnamic derivatives based on ferulic and caffeic acids were designed to meet the pharmacokinetic requirements to cross the blood-brain barrier and to display neuroprotective activity within the central nervous system. Biological screening included the assessment of acetylcholinesterase and glycogen synthase kinase 3β inhibition, iron chelation properties, in vitro blood-brain barrier permeability, evaluation of cytotoxicity and neuroprotection against 6-hydroxydopamine induced damage in SH-SY5Y cells. Although the chemical modifications did not significantly alter the in vitro activity of the parent compounds, the results of the PAMPA-BBB assay show that some derivatives have higher diffusion rates and may reach the brain. The majority of the synthesized compounds did not display cytotoxicity and successfully prevent 6-hydroxydopamine damage. In this series, compound 14 stands out as a promising neuroprotective agent combining a number of key features: iron chelation, neuroprotection against oxidative damage, mild acetylcholinesterase activity and ability to permeate the blood-brain barrier. This biology-oriented approach provides new tools for the generation of new chemical entities to tackle the oxidative damage associated with neurodegenerative disorders.Peer Reviewe

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-a), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-a-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-a-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-a-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-a 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-a 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-a and IL-1ß. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-a activation. In fact, both TNF-a-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-a-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.We would like to thank Dr. João Relvas laboratory for the help with N9 microglia cell culture; Dr. Sofia Lamas for the guidance on the animal welfare and support with animal experiments (Animal facility, i3S); and to LC Sciences for the miRNA microarray data and analysis. The mass spectrometry technique was performed by Hugo Osório at the i3S Proteomics Scientific Platform with support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01–0145-FEDER-022125). This work was funded by project NORTE-01–0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.P.B. and J.B. are supported by FCT–Fundação para a Ciência e Tecnologia, through BiotechHealth PhD program fellowship (PD/BD/135490/2018) and Areas of Basic and Applied Biology PhD program fellowship (PD/BD/135450/2017), respectively