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Schizophrenia is a mental disorder with sex bias in disease onset and symptom severity. Recently, it was observed that females present more severe symptoms in the perimenstrual phase of the menstrual cycle. The administration of estrogen also alleviates schizophrenia symptoms. Despite this, little is known about symptom fluctuation over the menstrual cycle and the underlying mechanisms. To address this issue, we worked with the two-hit schizophrenia animal model induced by neonatal exposure to a virus-like particle, Poly I:C, in association with peripubertal unpredictable stress exposure. Prepulse inhibition of the startle reflex (PPI) in male and female mice was considered analogous to human schizophrenia-like behavior. Female mice were studied in the proestrus (high-estrogen estrous cycle phase) and diestrus (low-estrogen phase). Additionally, we evaluated the hippocampal mRNA expression of estrogen synthesis proteins, TSPO and aromatase, and estrogen receptors ERα, ERβ, and GPER. We also collected Peripheral Blood Mononuclear Cells (PBMCs) from male and female patients with schizophrenia and converted them to induced microglia-like cells (iMGs) to evaluate the expression of GPER. We observed raised hippocampal expression of GPER in two-hit female mice at the proestrus phase without PPI deficits and higher levels of proteins related to estrogen synthesis, TSPO, and aromatase. In contrast, two-hit adult males with PPI deficits presented lower hippocampal mRNA expression of TSPO, aromatase, and GPER. iMGs from male and female patients with schizophrenia showed lower mRNA expression of GPER than controls. Therefore, our results suggest that GPER alterations constitute an underlying mechanism for sex influence in schizophrenia.

Amyloid deposits in Alzheimer’s disease (AD) are surrounded by large numbers of plaque-associated axonal spheroids (PAAS). PAAS disrupt axonal electrical conduction and neuronal network function, and correlate with AD severity. However, the mechanisms that govern their formation remain unknown. To uncover the molecular architecture of PAAS, we applied proximity labeling proteomics of spheroids in human AD postmortem brains and mice. We then implemented a human iPSC-derived AD model recapitulating PAAS pathology for mechanistic studies. Using this strategy, we uncovered hundreds of previously unknown PAAS-enriched proteins and signaling pathways, including PI3K/AKT/mTOR. Phosphorylated mTOR was highly enriched in PAAS and strongly correlated with disease severity in humans. Importantly, pharmacological mTOR inhibition in iPSC-derived human neurons or AAV-mediated knockdown in mice, led to a marked reduction of PAAS pathology. Altogether, our study provides a novel platform to examine mechanisms of axonal pathology in neurodegeneration and to evaluate the therapeutic potential of novel targets.

Synapses are well known as the main structures responsible for transmitting information through the release and recognition of neurotransmitters by pre- and post-synaptic neurons. These structures are widely formed and eliminated throughout the whole lifespan via processes termed synaptogenesis and synaptic pruning, respectively. Whilst the first process is needed for ensuring proper connectivity between brain regions and also with the periphery, the second phenomenon is important for their refinement by eliminating weaker and unnecessary synapses and, at the same time, maintaining and favoring the stronger ones, thus ensuring proper synaptic transmission. It is well-known that synaptic elimination is modulated by neuronal activity. However, only recently the role of the classical complement cascade in promoting this phenomenon has been demonstrated. Specifically, microglial cells recognize activated complement component 3 (C3) bound to synapses targeted for elimination, triggering their engulfment. As this is a highly relevant process for adequate neuronal functioning, disruptions or exacerbations in synaptic pruning could lead to severe circuitry alterations that could underlie neuropathological alterations typical of neurological and neuropsychiatric disorders. In this review, we focus on discussing the possible involvement of excessive synaptic elimination in Alzheimer’s disease, as it has already been reported dendritic spine loss in post-synaptic neurons, increased association of complement proteins with its synapses and, hence, augmented microglia-mediated pruning in animal models of this disorder. In addition, we briefly discuss how this phenomenon could be related to other neurological disorders, including multiple sclerosis and schizophrenia.

Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1β, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.

Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animal models, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human induced pluripotent stem cells (iPSC)-derived astrocytes which replicates aspects temporally intertwined of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, secretion of cytokines and changes in morphological phenotypes of human iPSC-derived astrocytes exposed to TNF-α. It was observed the NF-kB nuclear translocation, increases either in the inflammation-related cytokines secretion and gene expression for IL-1β, IL-6 and TNF-α following 24 h TNF-α stimulation. After 5 days, human iPSC-derived astrocytes exposed to TNF-α exhibited increases in vimentin and GFAP immunolabeling, elongated shape and shrinkage of nuclei, which is typical phenotypes of astrogliosis. Moreover, about a 50% decrease in D-[3H] aspartate uptake was observed over the astrogliosis course with no evident cell damage, which suggests astrocytic dysfunction. Taken together, our results indicate that cultured human iPSC-derived astrocytes reproduce canonical events associated to astrogliosis in a time dependent fashion. Our findings may contribute to a better understanding of mechanisms governing human astrogliosis. Furthermore, the approach described here presents a potential applicability as a platform to uncover novel biomarkers and new drug targets to refrain astrogliosis associated to human brain disorders.

Huntington’s disease (HD) is a neurodegenerative autosomal dominant disorder, characterized by symptoms of involuntary movement of the body, loss of cognitive function, psychiatric disorder, leading inevitably to death. It has been previously described that higher levels of brain expression of Cav1 channels are involved in major neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Our results demonstrate that a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD mice) at the age of 3 and 12 months exhibits significantly increased Cav1.2 protein levels in the cortex, as compared with wild-type littermates. Importantly, electrophysiological analyses confirm a significant increase in L-type Ca2+ currents and total Ca2+ current density in cortical neurons from BACHD mice. By using an in vitro assay to measure neuronal cell death, we were able to observe neuronal protection against glutamate toxicity after treatment with Cav1 blockers, in wild-type and, more importantly, in BACHD neurons. According to our data, Cav1 blockers may offer an interesting strategy for the treatment of HD. Altogether, our results show that mutant huntingtin (mHtt) expression may cause a dysregulation of Cav1.2 channels and we hypothesize that this contributes to neurodegeneration during HD.

A esquizofrenia é uma desordem psiquiátrica, cuja causa é uma conjunção de fatores genéticos, ambientais e do desenvolvimento. Os indivíduos com esta doença apresentam um quadro clínico caracterizado por sintomas positivos (psicose, alucinações e delírios), negativos (depressão, pensamento e fala desordenados e perda de interesse social) e déficits cognitivos, exibindo também uma redução no volume de massa cinzenta. Análises microanatômicas sugerem que a redução na massa cinzenta deve-se à diminuição na quantidade de espinhas dendríticas. A redução na quantidade destas estruturas em esquizofrênicos provavelmente deve-se a uma exacerbação do processo de eliminação sináptica que ocorre normalmente na adolescência e durante o início da idade adulta. Recentemente, foi elucidado que moléculas do sistema imunológico, como as vias clássica do sistema do complemento e de CX3CL1/CX3CR1, atuam diretamente neste processo, levando à fagocitose das sinapses imaturas pela microglia. Além disso, foi demonstrado que astrócitos secretam citocinas capazes de modular a via do complemento e promover a poda sináptica. Diante disso e tendo em vista que infecções pré-natais atuam como fatores de risco para esquizofrenia, este trabalho objetivou analisar a produção de citocinas e CX3CL1 nos astrócitos derivados de células-tronco de pluripotência induzida (hIPSCs) de indivíduos esquizofrênicos estimulados por TNF-α. Os resultados demonstram que a cinética de produção dos transcritos para TNF-α e IL-1β está alterada nos astrócitos de esquizofrênicos (SCZ) em relação aos astrócitos de indivíduos saudáveis (HC). Além disso, astrócitos SCZ produzem níveis basais aumentados de TGF-β3 e cinética de transcrição de CX3CL1 atrasada em relação aos HC. Finalmente, a forma não secretada de CX3CL1 parece estar aumentada nos astrócitos SCZ, sugerindo problemas na liberação da sua forma secretada. Embora estes resultados devam ser vistos com cautela diante da limitação do tamanho da amostra, eles indicam que os astrócitos dos indivíduos SCZ produzem imunomoduladores de forma distinta quando comparados a indivíduos saudáveis. Schizophrenia is a psychiatric disorder, caused by a conjunction of genetic, environmental and developmental factors. Individuals bearing this disease exhibit cognitive deficits, positive (psychosis, hallucinations and delusions) and negative symptoms (depression, disordered thoughts and speech and loss of social drive), as well as reduced gray matter volume. Microanatomical analyses suggest that this gray matter reduction is due to diminished dendritic spine density. This decrease in spine density observed in schizophrenic patients likely happens because of exaggerated synaptic elimination, a process that normally occurs during adolescence and early adulthood. Recently, it has been shown that immune molecules, such as components of the classical complement cascade and CX3CL1/CX3CR1 pathway, play a direct role in this process, triggering the phagocytosis of immature synapses by microglia. Moreover, it has been demonstrated that astrocytes secrete cytokines capable of modulating the complement cascade and promoting synaptic pruning. Taking into account that pre-natal infection acts as a risk factor for schizophrenia, this work aimed at analyzing cytokines and CX3CL1 production employing induced pluripotent stem cells (hIPSCs)-derived astrocytes from schizophrenic individuals after stimulation with TNF-α. The results demonstrate that TNF-α and IL-1β transcripts production kinetics are altered in schizophrenic-derived (SCZ) astrocytes relative to healthy control-derived (HC) astrocytes. Also, SCZ astrocytes produce increased basal levels of TGF-β3 and delayed CX3CL1 transcription kinetics relative to those of HC. Finally, non-secreted CX3CL1 levels seems to be increased in SCZ astrocytes, suggesting problems in the release of the soluble form. Even though these results should be regarded as preliminary due to the limited sample size, they clearly indicate that SCZ astrocytes produce immunomodulators in a distinct manner compared to HC astrocytes.

Zika virus is a mosquito-borne virus that is associated with neurodegenerative diseases, including Guillain–Barre syndrome1 and congenital Zika syndrome2. As Zika virus targets the nervous system, there is an urgent need to develop therapeutic strategies that inhibit Zika virus infection in the brain. Here, we have engineered a brain-penetrating peptide that works against Zika virus and other mosquito-borne viruses. We evaluated the therapeutic efficacy of the peptide in a lethal Zika virus mouse model exhibiting systemic and brain infection. Therapeutic treatment protected against mortality and markedly reduced clinical symptoms, viral loads and neuroinflammation, as well as mitigated microgliosis, neurodegeneration and brain damage. In addition to controlling systemic infection, the peptide crossed the blood–brain barrier to reduce viral loads in the brain and protected against Zika-virus-induced blood–brain barrier injury. Our findings demonstrate how engineering strategies can be applied to develop peptide therapeutics and support the potential of a brain-penetrating peptide to treat neurotropic viral infections. The Zika virus infects the central nervous system and results in severe brain malformation. An amphiphatic peptide is now shown to penetrate the blood–brain barrier, reducing viral loads due to its activity against Zika and other mosquito-borne viruses.

Exploiting the inhibition of host signaling pathways aiming for discovery of potential antiflaviviral compounds is clearly a beneficial strategy for the control of life-threatening diseases caused by flaviviruses. Here we describe the antiviral activity of the MEK1/2 inhibitor U0126 against Yellow fever virus 17D vaccine strain (YFV-17D). Infection of VERO cells with YFV-17D stimulates ERK1/2 phosphorylation early during infection. Pharmacological inhibition of MEK1/2 through U0126 treatment of VERO cells blockades not only the YFV-stimulated ERK1/2 phosphorylation, but also inhibits YFV replication by ∼99%. U0126 was also effective against dengue virus (DENV-2 and -3) and Saint-Louis encephalitis virus (SLEV). Levels of NS4AB, as detected by immunofluorescence, are diminished upon treatment with the inhibitor, as well as the characteristic endoplasmic reticulum membrane invagination stimulated during the infection. Though not protective, treatment of YFV-infected, adult BALB/c mice with U0126 resulted in significant reduction of virus titers in brains. Collectively, our data suggest the potential targeting of the MEK1/2 kinase as a therapeutic tool against diseases caused by flaviviruses such as yellow fever, adverse events associated with yellow fever vaccination and dengue.