Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+ , as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Metabolic switch in the aging astrocyte supported via integrative approach comprising network and transcriptome analyses.

Dysregulated central-energy metabolism is a hallmark of brain aging. Supplying enough energy for neurotransmission relies on the neuron-astrocyte metabolic network. To identify genes contributing to age-associated brain functional decline, we formulated an approach to analyze the metabolic network by integrating flux, network structure and transcriptomic databases of neurotransmission and aging. Our findings support that during brain aging: (1) The astrocyte undergoes a metabolic switch from aerobic glycolysis to oxidative phosphorylation, decreasing lactate supply to the neuron, while the neuron suffers intrinsic energetic deficit by downregulation of Krebs cycle genes, including mdh1 and mdh2 (Malate-Aspartate Shuttle); (2) Branched-chain amino acid degradation genes were downregulated, identifying dld as a central regulator; (3) Ketone body synthesis increases in the neuron, while the astrocyte increases their utilization, in line with neuronal energy deficit in favor of astrocytes. We identified candidates for preclinical studies targeting energy metabolism to prevent age-associated cognitive decline.

Quantitative Proteomics of Nervous System Regeneration: From Sample Preparation to Functional Data Analyses.

Mammals have a limited regenerative capacity, especially of the central nervous system. Consequently, any traumatic injury or neurodegenerative disease results in irreversible damage. An important approach to finding strategies to promote regeneration in mammals has been the study of regenerative organisms like Xenopus, the axolotl, and teleost fish. High-throughput technologies like RNA-Seq and quantitative proteomics are starting to provide valuable insight into the molecular mechanisms that drive nervous system regeneration in these organisms. In this chapter, we present a detailed protocol for performing iTRAQ proteomics that can be applied to the analysis of nervous system samples, using Xenopus laevis as an example. The quantitative proteomics protocol and directions for performing functional enrichment data analyses of gene lists (e.g., differentially abundant proteins from a proteomic study, or any type of high-throughput analysis) are aimed at the general bench biologist and do not require previous programming knowledge.

Cornifelin expression during Xenopus laevis metamorphosis and in response to spinal cord injury.

BACKGROUND: In a high-throughput RNA sequencing analysis, comparing the transcriptional response between Xenopus laevis regenerative and non-regenerative stages to spinal cord injury, cornifelin was found among the most highly differentially expressed genes. Cornifelin is mainly expressed in stratified squamous epithelia, but its expression in the spinal cord and other central nervous structures has only been described during early development. RESULTS: Here, we report cornifelin expression in the spinal cord, retina, and cornea throughout metamorphosis and in the spinal cord after injury. Cornifelin was detected in the grey matter and meninges of the spinal cord from NF-50 to NF-66, with decreased expression in the grey matter during metamorphosis. In the retina, cornifelin was expressed in the ganglion cell layer, the inner and outer nuclear layer, and the outer segment from NF-50 to NF-66. After spinal cord injury, we only observed cornifelin upregulation in NF-66 but no significant changes in NF-50. However, we found cornifelin positive cells in NF-50 meninges closing the spinal cord stumps 1 day after injury and delineating the borders of the spinal cord following the continuity of tissue regeneration in the following days after injury. Instead, in NF-66, cornifelin positive cells were distributed to the ventral side of the spinal cord at 6 days after injury, and at the injury gap at 10 days after injury. CONCLUSIONS: Cornifelin is expressed in the Xenopus laevis spinal cord and eye during metamorphosis and plays a role in the meningeal response to spinal cord injury.

Neuron-intrinsic origin of hyperexcitability during early pathogenesis of Alzheimer's disease: An Editorial Highlight for "Hippocampal hyperactivity in a rat model of Alzheimer's disease" on https://doi.org/10.1111/jnc.15323.

In Alzheimer's disease (AD), hippocampal hyperactivation is already present at early stages of the disorder, in some cases, even when the individual is still asymptomatic. Neuronal hyperexcitability has been described to occur before the deposition of amyloid beta plaques in mouse models of AD and has been attributed to an imbalance between excitatory and inhibitory activity. In this Editorial Highlight, we discuss the article by Sosulina et al., published in this issue of the Journal of Neurochemistry, which offers novel insights into the possible origins of this neuronal excitability observed during the early pathogenesis of AD.

Neurod4 converts endogenous neural stem cells to neurons with synaptic formation after spinal cord injury.

The transcriptome analysis of injured Xenopus laevis tadpole and mice suggested that Neurod4L.S., a basic-helix-loop-helix transcription factor, was the most promising transcription factor to exert neuroregeneration after spinal cord injury (SCI) in mammals. We generated a pseudotyped retroviral vector with the neurotropic lymphocytic choriomeningitis virus (LCMV) envelope to deliver murine Neurod4 to mice undergoing SCI. SCI induced ependymal cells to neural stem cells (NSCs) in the central canal. The LCMV envelope-based pseudotypedvector preferentially introduced Neurod4 into activated NSCs, which converted to neurons with axonal regrowth and suppressed the scar-forming glial lineage. Neurod4-induced inhibitory neurons predominantly projected to the subsynaptic domains of motor neurons at the epicenter, and Neurod4-induced excitatory neurons predominantly projected to subsynaptic domains of motor neurons caudal to the injury site suggesting the formation of functional synapses. Thus, Neurod4 is a potential therapeutic factor that can improve anatomical and functional recovery after SCI.

Quantitative Proteomics After Spinal Cord Injury (SCI) in a Regenerative and a Nonregenerative Stage in the Frog Xenopus laevis.

The capacity to regenerate the spinal cord after an injury is a coveted trait that only a limited group of nonmammalian organisms can achieve. In Xenopus laevis, this capacity is only present during larval or tadpole stages, but is absent during postmetamorphic frog stages. This provides an excellent model for comparative studies between a regenerative and a nonregenerative stage to identify the cellular and molecular mechanisms that explain this difference in regenerative potential. Here, we used iTRAQ chemistry to obtain a quantitative proteome of the spinal cord 1 day after a transection injury in regenerative and nonregenerative stage animals, and used sham operated animals as controls. We quantified a total of 6,384 proteins, with 172 showing significant differential expression in the regenerative stage and 240 in the nonregenerative stage, with an overlap of only 14 proteins. Functional enrichment analysis revealed that although the regenerative stage downregulated synapse/vesicle and mitochondrial proteins, the nonregenerative stage upregulated lipid metabolism proteins, and downregulated ribosomal and translation control proteins. Furthermore, STRING network analysis showed that proteins belonging to these groups are highly interconnected, providing interesting candidates for future functional studies. Data are available via ProteomeXchange with identifier PXD006993.

Spinal cord regeneration in Xenopus laevis.

Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1-2 months; SCI can be achieved in 5-15 min; and swimming recovery takes 20-30 d.

The African clawed frog Xenopus laevis: A model organism to study regeneration of the central nervous system.

While an injury to the central nervous system (CNS) in humans and mammals is irreversible, amphibians and teleost fish have the capacity to fully regenerate after severe injury to the CNS. Xenopus laevis has a high potential to regenerate the brain and spinal cord during larval stages (47-54), and loses this capacity during metamorphosis. The optic nerve has the capacity to regenerate throughout the frog's lifespan. Here, we review CNS regeneration in frogs, with a focus in X. laevis, but also provide some information about X. tropicalis and other frogs. We start with an overview of the anatomy of the Xenopus CNS, including the main supraspinal tracts that emerge from the brain stem, which play a key role in motor control and are highly conserved across vertebrates. We follow with the advantages of using Xenopus, a classical laboratory model organism, with increasing availability of genetic tools like transgenesis and genome editing, and genomic sequences for both X. laevis and X. tropicalis. Most importantly, Xenopus provides the possibility to perform intra-species comparative experiments between regenerative and non-regenerative stages that allow the identification of which factors are permissive for neural regeneration, and/or which are inhibitory. We aim to provide sufficient evidence supporting how useful Xenopus can be to obtain insights into our understanding of CNS regeneration, which, complemented with studies in mammalian vertebrate model systems, can provide a collaborative road towards finding novel therapeutic approaches for injuries to the CNS.

Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages.

BACKGROUND: Xenopus laevis has regenerative and non-regenerative stages. As a tadpole, it is fully capable of functional recovery after a spinal cord injury, while its juvenile form (froglet) loses this capability during metamorphosis. We envision that comparative studies between regenerative and non-regenerative stages in Xenopus could aid in understanding why spinal cord regeneration fails in human beings. RESULTS: To identify the mechanisms that allow the tadpole to regenerate and inhibit regeneration in the froglet, we obtained a transcriptome-wide profile of the response to spinal cord injury in Xenopus regenerative and non-regenerative stages. We found extensive transcriptome changes in regenerative tadpoles at 1 day after injury, while this was only observed by 6 days after injury in non-regenerative froglets. In addition, when comparing both stages, we found that they deployed a very different repertoire of transcripts, with more than 80% of them regulated in only one stage, including previously unannotated transcripts. This was supported by gene ontology enrichment analysis and validated by RT-qPCR, which showed that transcripts involved in metabolism, response to stress, cell cycle, development, immune response and inflammation, neurogenesis, and axonal regeneration were regulated differentially between regenerative and non-regenerative stages. CONCLUSIONS: We identified differences in the timing of the transcriptional response and in the inventory of regulated transcripts and biological processes activated in response to spinal cord injury when comparing regenerative and non-regenerative stages. These genes and biological processes provide an entry point to understand why regeneration fails in mammals. Furthermore, our results introduce Xenopus laevis as a genetic model organism to study spinal cord regeneration.

Spinal cord regeneration: lessons for mammals from non-mammalian vertebrates.

Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative-permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so.

Transcriptomics using next generation sequencing technologies.

Next generation sequencing technologies may now be applied to the study of transcriptomics. RNA-Seq or RNA sequencing employs high-throughput sequencing of complementary DNA fragments delivering a transcriptional profile. In this chapter, we aim to provide a starting point for Xenopus researchers planning on starting an RNA-Seq transcriptomics study. We begin by providing a section on template isolation and library preparation. The next section comprises the main bioinformatics procedures that need to be performed for raw data processing, normalization, and differential gene expression. Finally, we have included a section on studying deep sequencing results in Xenopus, which offers general guidance as to what can be done in this model.

Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells.

BACKGROUND: In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. RESULTS: Here, we report that tail amputation results in a global increase of Sox2 levels and proliferation of Sox2(+) cells. Overexpression of a dominant negative form of Sox2 diminished proliferation of spinal cord resident cells affecting tail regeneration after amputation, suggesting that spinal cord regeneration is crucial for the whole process. After spinal cord transection, Sox2(+) cells are found in the ablation gap forming aggregates. Furthermore, Sox2 levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2 levels at non-regenerative stages. CONCLUSIONS: Sox2(+) cells contribute to the regeneration of spinal cord after tail amputation and transection. Sox2 levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord damage activates proliferation and/or migration of Sox2(+) cells, thus allowing regeneration of the spinal cord after tail amputation or reconstitution of the ependymal epithelium after spinal cord transection.

Expression of transposable elements in neural tissues during Xenopus development.

Transposable elements comprise a large proportion of animal genomes. Transposons can have detrimental effects on genome stability but also offer positive roles for genome evolution and gene expression regulation. Proper balance of the positive and deleterious effects of transposons is crucial for cell homeostasis and requires a mechanism that tightly regulates their expression. Herein we describe the expression of DNA transposons of the Tc1/mariner superfamily during Xenopus development. Sense and antisense transcripts containing complete Tc1-2_Xt were detected in Xenopus embryos. Both transcripts were found in zygotic stages and were mainly localized in Spemann's organizer and neural tissues. In addition, the Tc1-like elements Eagle, Froggy, Jumpy, Maya, Xeminos and TXr were also expressed in zygotic stages but not oocytes in X. tropicalis. Interestingly, although Tc1-2_Xt transcripts were not detected in Xenopus laevis embryos, transcripts from other two Tc1-like elements (TXr and TXz) presented a similar temporal and spatial pattern during X. laevis development. Deep sequencing analysis of Xenopus tropicalis gastrulae showed that PIWI-interacting RNAs (piRNAs) are specifically derived from several Tc1-like elements. The localized expression of Tc1-like elements in neural tissues suggests that they could play a role during the development of the Xenopus nervous system.

Expression of DNA transposable elements during nervous system development: A discussion about its possible functions.

Transposable elements (retrotransposons and DNA transposons) comprise a large proportion of animal genomes, for example 20% in D. melanogaster, 36% in X. tropicalis and 45% in humans. After invading a new genome, the transposable element increases its copy number and subsequently accumulates mutations. These may eventually result in inactive copies. Until recent days transposons have been considered "junk" DNA and no clear function have been assigned for this important amount of information on genomes.

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole.

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor beta and PGE(2) activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE(2) synthesis inhibition. Nevertheless, NO() levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.