Diving into the proteomic atlas of SARS-CoV-2 infected cells.

The COVID-19 pandemic was initiated by the rapid spread of a SARS-CoV-2 strain. Though mainly classified as a respiratory disease, SARS-CoV-2 infects multiple tissues throughout the human body, leading to a wide range of symptoms in patients. To better understand how SARS-CoV-2 affects the proteome from cells with different ontologies, this work generated an infectome atlas of 9 cell models, including cells from brain, blood, digestive system, and adipocyte tissue. Our data shows that SARS-CoV-2 infection mainly trigger dysregulations on proteins related to cellular structure and energy metabolism. Despite these pivotal processes, heterogeneity of infection was also observed, highlighting many proteins and pathways uniquely dysregulated in one cell type or ontological group. These data have been made searchable online via a tool that will permit future submissions of proteomic data ( https://reisdeoliveira.shinyapps.io/Infectome_App/ ) to enrich and expand this knowledgebase.

NMDA glutamate receptor antagonist MK-801 induces proteome changes in adult human brain slices which are partially counteracted by haloperidol and clozapine.

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.

Induced-pluripotent stem cells and neuroproteomics as tools for studying neurodegeneration.

The investigation of neurodegenerative diseases advanced significantly with the advent of cell-reprogramming technology, leading to the creation of new models of human illness. These models, derived from induced pluripotent stem cells (iPSCs), facilitate the study of sporadic as well as hereditary diseases and provide a comprehensive understanding of the molecular mechanisms involved with neurodegeneration. Through proteomics, a quantitative tool capable of identifying thousands of proteins from small sample volumes, researchers have attempted to identify disease mechanisms by detecting differentially expressed proteins and proteoforms in disease models, biofluids, and postmortem brain tissue. The integration of these two technologies allows for the identification of novel pathological targets within the realm of neurodegenerative diseases. Here, we highlight studies from the past 5 years on the contributions of iPSCs within neuroproteomic investigations, which uncover the molecular mechanisms behind these illnesses.

Gut microbiota from patients with COVID-19 cause alterations in mice that resemble post-COVID symptoms.

Long-term sequelae of coronavirus disease (COVID)-19 are frequent and of major concern. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects the host gut microbiota, which is linked to disease severity in patients with COVID-19. Here, we report that the gut microbiota of post-COVID subjects had a remarkable predominance of Enterobacteriaceae strains with an antibiotic-resistant phenotype compared to healthy controls. Additionally, short-chain fatty acid (SCFA) levels were reduced in feces. Fecal transplantation from post-COVID subjects to germ-free mice led to lung inflammation and worse outcomes during pulmonary infection by multidrug-resistant Klebsiella pneumoniae. transplanted mice also exhibited poor cognitive performance. Overall, we show prolonged impacts of SARS-CoV-2 infection on the gut microbiota that persist after subjects have cleared the virus. Together, these data demonstrate that the gut microbiota can directly contribute to post-COVID sequelae, suggesting that it may be a potential therapeutic target.

Mitochondrial, cell cycle control and neuritogenesis alterations in an iPSC-based neurodevelopmental model for schizophrenia.

Schizophrenia is a severe psychiatric disorder of neurodevelopmental origin that affects around 1% of the world's population. Proteomic studies and other approaches have provided evidence of compromised cellular processes in the disorder, including mitochondrial function. Most of the studies so far have been conducted on postmortem brain tissue from patients, and therefore, do not allow the evaluation of the neurodevelopmental aspect of the disorder. To circumvent that, we studied the mitochondrial and nuclear proteomes of neural stem cells (NSCs) and neurons derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients versus healthy controls to assess possible alterations related to energy metabolism and mitochondrial function during neurodevelopment in the disorder. Our results revealed differentially expressed proteins in pathways related to mitochondrial function, cell cycle control, DNA repair and neuritogenesis and their possible implication in key process of neurodevelopment, such as neuronal differentiation and axonal guidance signaling. Moreover, functional analysis of NSCs revealed alterations in mitochondrial oxygen consumption in schizophrenia-derived cells and a tendency of higher levels of intracellular reactive oxygen species (ROS). Hence, this study shows evidence that alterations in important cellular processes are present during neurodevelopment and could be involved with the establishment of schizophrenia, as well as the phenotypic traits observed in adult patients. Neural stem cells (NSCs) and neurons were derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients and controls. Proteomic analyses were performed on the enriched mitochondrial and nuclear fractions of NSCs and neurons. Whole-cell proteomic analysis was also performed in neurons. Our results revealed alteration in proteins related to mitochondrial function, cell cycle control, among others. We also performed energy pathway analysis and reactive oxygen species (ROS) analysis of NSCs, which revealed alterations in mitochondrial oxygen consumption and a tendency of higher levels of intracellular ROS in schizophrenia-derived cells.

Exogenous succinate impacts mouse brown adipose tissue mitochondrial proteome and potentiates body mass reduction induced by liraglutide.

Obesity is one of the leading noncommunicable diseases in the world. Despite intense efforts to develop strategies to prevent and treat obesity, its prevalence continues to rise worldwide. A recent study has shown that the tricarboxylic acid intermediate succinate increases body energy expenditure by promoting brown adipose tissue thermogenesis through the activation of uncoupling protein-1; this has generated interest surrounding its potential usefulness as an approach to treat obesity. It is currently unknown how succinate impacts brown adipose tissue protein expression, and how exogenous succinate impacts body mass reduction promoted by a drug approved to treat human obesity, the glucagon-like-1 receptor agonist, liraglutide. In the first part of this study, we used bottom-up shotgun proteomics to determine the acute impact of exogenous succinate on the brown adipose tissue. We show that succinate rapidly affects the expression of 177 brown adipose tissue proteins, which are mostly associated with mitochondrial structure and function. In the second part of this study, we performed a short-term preclinical pharmacological intervention, treating diet-induced obese mice with a combination of exogenous succinate and liraglutide. We show that the combination was more efficient than liraglutide alone in promoting body mass reduction, food energy efficiency reduction, food intake reduction, and an increase in body temperature. Using serum metabolomics analysis, we showed that succinate, but not liraglutide, promoted a significant increase in the blood levels of several medium and long-chain fatty acids. In conclusion, exogenous succinate promotes rapid changes in brown adipose tissue mitochondrial proteins, and when used in association with liraglutide, increases body mass reduction.NEW & NOTEWORTHY Exogenous succinate induces major changes in brown adipose tissue protein expression affecting particularly mitochondrial respiration and structural proteins. When given exogenously in drinking water, succinate mitigates body mass gain in a rodent model of diet-induced obesity; in addition, when given in association with the glucagon-like peptide-1 receptor agonist, liraglutide, succinate increases body mass reduction promoted by liraglutide alone.

Proteomic signatures of schizophrenia-sourced iPSC-derived neural cells and brain organoids are similar to patients' postmortem brains.

BACKGROUND: Schizophrenia is a complex and severe neuropsychiatric disorder, with a wide range of debilitating symptoms. Several aspects of its multifactorial complexity are still unknown, and some are accepted to be an early developmental deficiency with a more specifically neurodevelopmental origin. Understanding the timepoints of disturbances during neural cell differentiation processes could lead to an insight into the development of the disorder. In this context, human brain organoids and neural cells differentiated from patient-derived induced pluripotent stem cells are of great interest as a model to study the developmental origins of the disease. RESULTS: Here we evaluated the differential expression of proteins of schizophrenia patient-derived neural progenitors (NPCs), early neurons, and brain organoids in comparison to healthy individuals. Using bottom-up shotgun proteomics with a label-free approach for quantitative analysis, we found multiple dysregulated proteins since NPCs, modified, and disrupted the 21DIV neuronal differentiation, and cerebral organoids. Our experimental methods have shown impairments in pathways never before found in patient-derived induced pluripotent stem cells studies, such as spliceosomes and amino acid metabolism; but also, those such as axonal guidance and synaptogenesis, in line with postmortem tissue studies of schizophrenia patients. CONCLUSION: In conclusion, here we provide comprehensive, large-scale, protein-level data of different neural cell models that may uncover early events in brain development, underlying several of the mechanisms within the origins of schizophrenia.

Protein Succinylation and Malonylation as Potential Biomarkers in Schizophrenia.

Two protein post-translational modifications, lysine succinylation and malonylation, are implicated in protein regulation, glycolysis, and energy metabolism. The precursors of these modifications, succinyl-CoA and malonyl-CoA, are key players in central metabolic processes. Both modification profiles have been proven to be responsive to metabolic stimuli, such as hypoxia. As mitochondrial dysfunction and metabolic dysregulation are implicated in schizophrenia and other psychiatric illnesses, these modification profiles have the potential to reveal yet another layer of protein regulation and can furthermore represent targets for biomarkers that are indicative of disease as well as its progression and treatment. In this work, data from shotgun mass spectrometry-based quantitative proteomics were compiled and analyzed to probe the succinylome and malonylome of postmortem brain tissue from patients with schizophrenia against controls and the human oligodendrocyte precursor cell line MO3.13 with the dizocilpine chemical model for schizophrenia, three antipsychotics, and co-treatments. Several changes in the succinylome and malonylome were seen in these comparisons, revealing these modifications to be a largely under-studied yet important form of protein regulation with broad potential applications.

Modeling Schizophrenia In Vitro: Challenges and Insights on Studying Brain Cells.

One of the challenges in studying neuropsychiatric disorders is the difficulty in accessing brain tissue from living patients. Schizophrenia is a chronic mental illness that affects 1% of the population worldwide, and its development stems from genetic and environmental factors. In order to better understand the pathophysiology underlying schizophrenia, the development of efficient in vitro methods to model this disorder has been required. In addition to several in vitro models, induced pluripotent stem cells (iPSCs) arose as a powerful tool, enabling access to the genetic background of the donor. Moreover, genetic modification of these cells can improve studies of specific dysfunctions observed in the pathophysiology of several neuropsychiatric disorders, not only schizophrenia. Here, we summarize which in vitro models are currently available and their applications in schizophrenia research, describing their advantages and limitations. These technologies in the cell culture field hold great potential to contribute to a better understanding of the pathophysiology of schizophrenia in an integrated manner, in addition to testing potential therapeutic interventions based on the genetic background of the patient.

What Can We Learn from Animal Models to Study Schizophrenia?

Schizophrenia is a complex and heterogeneous neurodevelopmental psychiatric disorder characterized by a variety of symptoms classically grouped into three main domains: positive (hallucinations, delusions, and thought disorder) and negative symptoms (social withdrawal, lack of affect) and cognitive dysfunction (attention, working and episodic memory functions, and processing speed). This disorder places an immense emotional and economic pressure on the individual and society-at-large. Although the etiology of schizophrenia is not completely known, it is proposed to involve abnormalities in neurodevelopmental processes and dysregulation in the signaling mediated by several neurotransmitters, such as dopamine, glutamate, and GABA. Preclinical research using animal models are essential in our understanding of disease development and pathology as well as the discovery and advance of novel treatment choices. Here we describe rodent models for studying schizophrenia, including those based on the effects of drugs (pharmacological models), neurodevelopmental disruption, demyelination, and genetic alterations. The advantages and limitations of such models are highlighted. We also discussed the great potential of proteomic technologies in unraveling the molecular mechanism of schizophrenia through animal models.

Molecular Findings Guiding the Modulation of the Endocannabinoid System as a Potential Target to Treat Schizophrenia.

Schizophrenia is a psychiatric disorder of neurodevelopmental origin that is thought to result from the combination of genetic and socioenvironmental factors. Several studies have linked the endocannabinoid system with the pathophysiology of schizophrenia. Here, we provide a brief overview of the role of the endocannabinoid system (ECS) in the context of biological processes relevant to schizophrenia, such as neurodevelopment, synaptic plasticity, and brain energy metabolism. We also discuss alterations related to the ECS in schizophrenia and current efforts in both in vivo and in vitro studies that have provided a better understanding of the functioning of this system in the context of the disorder. Finally, we highlighted the modulation of the ECS as a potential for discovering novel therapeutic targets, suggesting new avenues for future research in the field.

Mitochondrial Dysregulation and the Influence in Neurodegenerative Diseases.

Mitochondrial function is essential to ensure vital cellular processes. Given the energy requirement of the brain, neuronal function, viability, and survival are closely related to proper mitochondrial function. Dysregulation of mitochondrial processes can lead to several detrimental effects in the cells and stablish the condition of mitochondrial dysfunction. This dysfunction is proposed to be greatly implicated in several neurodegenerative diseases, with evidence of compromised mitochondrial function and dynamics in Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

Morphological, cellular, and molecular basis of brain infection in COVID-19 patients.

Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. We show the spectrum of cerebral impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, ranging from long-term alterations in mildly infected individuals (orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms) to severe acute damage confirmed in brain tissue samples extracted from the orbitofrontal region (via endonasal transethmoidal access) from individuals who died of COVID-19. In an independent cohort of 26 individuals who died of COVID-19, we used histopathological signs of brain damage as a guide for possible SARS-CoV-2 brain infection and found that among the 5 individuals who exhibited those signs, all of them had genetic material of the virus in the brain. Brain tissue samples from these five patients also exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a noncanonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that reduces neuronal viability. Our data support the model in which SARS-CoV-2 reaches the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients.

Zika Virus Strains and Dengue Virus Induce Distinct Proteomic Changes in Neural Stem Cells and Neurospheres.

Brain abnormalities and congenital malformations have been linked to the circulating strain of Zika virus (ZIKV) in Brazil since 2016 during the microcephaly outbreak; however, the molecular mechanisms behind several of these alterations and differential viral molecular targets have not been fully elucidated. Here we explore the proteomic alterations induced by ZIKV by comparing the Brazilian (Br ZIKV) and the African (MR766) viral strains, in addition to comparing them to the molecular responses to the Dengue virus type 2 (DENV). Neural stem cells (NSCs) derived from induced pluripotent stem (iPSCs) were cultured both as monolayers and in suspension (resulting in neurospheres), which were then infected with ZIKV (Br ZIKV or ZIKV MR766) or DENV to assess alterations within neural cells. Large-scale proteomic analyses allowed the comparison not only between viral strains but also regarding the two- and three-dimensional cellular models of neural cells derived from iPSCs, and the effects on their interaction. Altered pathways and biological processes were observed related to cell death, cell cycle dysregulation, and neurogenesis. These results reinforce already published data and provide further information regarding the biological alterations induced by ZIKV and DENV in neural cells.

Cannabinoids modulate proliferation, differentiation, and migration signaling pathways in oligodendrocytes.

Cannabinoid signaling, mainly via CB1 and CB2 receptors, plays an essential role in oligodendrocyte health and functions. However, the specific molecular signals associated with the activation or blockade of CB1 and CB2 receptors in this glial cell have yet to be elucidated. Mass spectrometry-based shotgun proteomics and in silico biology tools were used to determine which signaling pathways and molecular mechanisms are triggered in a human oligodendrocytic cell line (MO3.13) by several pharmacological stimuli: the phytocannabinoid cannabidiol (CBD); CB1 and CB2 agonists ACEA, HU308, and WIN55, 212-2; CB1 and CB2 antagonists AM251 and AM630; and endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The modulation of cannabinoid signaling in MO3.13 was found to affect pathways linked to cell proliferation, migration, and differentiation of oligodendrocyte progenitor cells. Additionally, we found that carbohydrate and lipid metabolism, as well as mitochondrial function, were modulated by these compounds. Comparing the proteome changes and upstream regulators among treatments, the highest overlap was between the CB1 and CB2 antagonists, followed by overlaps between AEA and 2-AG. Our study opens new windows of opportunities, suggesting that cannabinoid signaling in oligodendrocytes might be relevant in the context of demyelinating and neurodegenerative diseases. Proteomics data are available at ProteomeXchange (PXD031923).

The Penicillium brasilianum Histone Deacetylase Clr3 Regulates Secondary Metabolite Production and Tolerance to Oxidative Stress.

Most of the biosynthetic gene clusters (BGCs) found in microbes are silent under standard laboratory cultivation conditions due to the lack of expression triggering stimuli, representing a considerable drawback in drug discovery. To access the full biosynthetic potential, studies towards the activation of cryptic BGCs are essential. Histone acetylation status is an important regulator of chromatin structure, which impacts cell physiology and the expression of BGCs. In this study, clr3, a gene encoding a histone deacetylase in Penicillium brasilianum LaBioMMi 136, is deleted and associated phenotypic and metabolic changes are evaluated. The results indicate reduced growth under oxidative stress conditions in the ∆clr3 strain, higher intracellular reactive oxygen species (ROS) levels, and a different transcriptional profile of 13 ROS-related genes of both strains under basal and ROS-induced conditions. Moreover, the production of 14 secondary metabolites, including austin-related meroterpenoids, brasiliamides, verruculogen, penicillic acid, and cyclodepsipeptides was evaluated in the ∆clr3 strain, most of them being reduced. Accordingly, the addition of epigenetic modulators responsible for HDAC inhibition into P. brasilianum's growth media also culminated in the reduction in secondary metabolite production. The results suggest that Clr3 plays an essential role in secondary metabolite biosynthesis in P. brasilianum, thus offering new strategies for the regulation of natural product synthesis by assessing chromatin modification.

Linking proteomic alterations in schizophrenia hippocampus to NMDAr hypofunction in human neurons and oligodendrocytes.

Glutamatergic neurotransmission dysfunction and the early involvement of the hippocampus have been proposed to be important aspects of the pathophysiology of schizophrenia. Here, we performed proteomic analysis of hippocampus postmortem samples from schizophrenia patients as well as neural cells-neurons and oligodendrocytes-treated with MK-801, an NMDA receptor antagonist. There were similarities in processes such as oxidative stress and apoptotic process when comparing hippocampus samples with MK-801-treated neurons, and in proteins synthesis when comparing hippocampus samples with MK-801-treated oligodendrocytes. This reveals that studying the effects of glutamatergic dysfunction in different neural cells can contribute to a better understanding of what it is observed in schizophrenia patients' postmortem brains.

Effects on Glial Cell Glycolysis in Schizophrenia: An Advanced Aging Phenotype?

Schizophrenia is a severe and debilitating psychiatric disorder believed to have neurodevelopmental origins. Several studies have associated energy metabolism dysfunction with the disorder, mostly related to glycolysis alterations. Glucose is the obligatory energy substrate of the brain and glycolysis is the first step for its metabolism. This takes place predominantly in glial cells, astrocytes and oligodendrocytes, whereas neurons present a predominant oxidative profile. Thus, glial cells generate either lactate or pyruvate to neurons for ATP production. In addition, some aspects of schizophrenia may reflect an advanced aging phenotype with effects on various neural cell types at different stages of the disease. Given the role of glial cells in brain energy metabolism, the association of glycolysis dysfunction and the accelerated aging of neuronal cells in schizophrenia, studies focusing on those aspects can yield important insights into the causes and implications of the disorder. In turn, this may lead to novel therapeutic strategies for improved treatment of individuals suffering with this disorder.

Synapse preservation and decreased glial reactions following ventral root crush (VRC) and treatment with 4-hydroxy-tempo (TEMPOL).

Astrogliosis and microglial reactions are correlated with the formation of scar tissue and synapse loss. 4-hydroxy-tempo (TEMPOL) is a reactive oxygen species scavenger with proven neuroprotective efficacy in experimental models of traumatic injury and cerebral ischemia. TEMPOL has not, however, been applied following ventral root lesions, which are particularly correlated with the degeneration of spinal motoneurons following brachial plexus injuries. Thus, the present study investigated the effects of TEMPOL on motoneurons and adjacent glial reactions, with a particular focus on the preservation of excitatory and inhibitory spinal circuits. Adult female Sprague Dawley rats were subjected to ventral root crush (VRC) at the lumbar intumescence. Animals were divided into the following experimental groups: (a) VRC-saline treatment; (b) VRC-TEMPOL treatment (12 mg/kg, n = 5), and (c) VRC-TEMPOL treatment (250 mg/kg, n = 5). The spinal cord tissue located contralateral to the lesion was used as the control. Fourteen days after lesioning, the rats were euthanized and the spinal cords were removed for motoneuron counting and immunolabeling with glial (GFAP and Iba-1) and synapse markers (synaptophysin, VGLUT-1, and GAD65). Although TEMPOL did not exert neuroprotective effects at the studied concentrations, the modulation of glial reactions was significant at higher doses. Thus, synaptophysin staining was preserved and, in particular, VGLUT-1-positive inputs were maintained, thereby indicating that TEMPOL preserved proprioceptive glutamatergic inputs without exacerbating the rate of motoneuron degeneration. Consequently, its administration with other efficient neuroprotective substances may significantly improve the outcomes following spinal cord lesioning.

The Energy Metabolism Dysfunction in Psychiatric Disorders Postmortem Brains: Focus on Proteomic Evidence.

Psychiatric disorders represent a great medical and social challenge and people suffering from these conditions face many impairments regarding personal and professional life. In addition, a mental disorder will manifest itself in approximately one quarter of the world's population at some period of their life. Dysfunction in energy metabolism is one of the most consistent scientific findings associated with these disorders. With this is mind, this review compiled data on disturbances in energy metabolism found by proteomic analyses of postmortem brains collected from patients affected by the most prevalent psychiatric disorders: schizophrenia (SCZ), bipolar disorder (BPD), and major depressive disorder (MDD). We searched in the PubMed database to gather the studies and compiled all the differentially expressed proteins reported in each work. SCZ studies revealed 92 differentially expressed proteins related to energy metabolism, while 95 proteins were discovered in BPD, and 41 proteins in MDD. With the compiled data, it was possible to determine which proteins related to energy metabolism were found to be altered in all the disorders as well as which ones were altered exclusively in one of them. In conclusion, the information gathered in this work could contribute to a better understanding of the impaired metabolic mechanisms and hopefully bring insights into the underlying neuropathology of psychiatric disorders.