Mitochondrial modulation with leriglitazone as a potential treatment for Rett syndrome.

BACKGROUND: Rett syndrome is a neuropediatric disease occurring due to mutations in MECP2 and characterized by a regression in the neuronal development following a normal postnatal growth, which results in the loss of acquired capabilities such as speech or purposeful usage of hands. While altered neurotransmission and brain development are the center of its pathophysiology, alterations in mitochondrial performance have been previously outlined, shaping it as an attractive target for the disease treatment. METHODS: We have thoroughly described mitochondrial performance in two Rett models, patients' primary fibroblasts and female Mecp2tm1.1Bird-/+ mice brain, discriminating between different brain areas. The characterization was made according to their bioenergetics function, oxidative stress, network dynamics or ultrastructure. Building on that, we have studied the effect of leriglitazone, a PPARγ agonist, in the modulation of mitochondrial performance. For that, we treated Rett female mice with 75 mg/kg/day leriglitazone from weaning until sacrifice at 7 months, studying both the mitochondrial performance changes and their consequences on the mice phenotype. Finally, we studied its effect on neuroinflammation based on the presence of reactive glia by immunohistochemistry and through a cytokine panel. RESULTS: We have described mitochondrial alterations in Rett fibroblasts regarding both shape and bioenergetic functions, as they displayed less interconnected and shorter mitochondria and reduced ATP production along with increased oxidative stress. The bioenergetic alterations were recalled in Rett mice models, being especially significant in cerebellum, already detectable in pre-symptomatic stages. Treatment with leriglitazone recovered the bioenergetic alterations both in Rett fibroblasts and female mice and exerted an anti-inflammatory effect in the latest, resulting in the amelioration of the mice phenotype both in general condition and exploratory activity. CONCLUSIONS: Our studies confirm the mitochondrial dysfunction in Rett syndrome, setting the differences through brain areas and disease stages. Its modulation through leriglitazone is a potential treatment for this disorder, along with other diseases with mitochondrial involvement. This work constitutes the preclinical necessary evidence to lead to a clinical trial.

Unraveling Molecular Pathways Altered in MeCP2-Related Syndromes, in the Search for New Potential Avenues for Therapy.

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked epigenetic modulator whose dosage is critical for neural development and function. Loss-of-function mutations in MECP2 cause Rett Syndrome (RTT, OMIM #312750) while duplications in the Xq28 locus containing MECP2 and Interleukin-1 receptor-associated kinase 1 (IRAK1) cause MECP2 duplication syndrome (MDS, OMIM #300260). Both are rare neurodevelopmental disorders that share clinical symptoms, including intellectual disability, loss of speech, hand stereotypies, vasomotor deficits and seizures. The main objective of this exploratory study is to identify novel signaling pathways and potential quantitative biomarkers that could aid early diagnosis and/or the monitoring of disease progression in clinical trials. We analyzed by RT-PCR gene expression in whole blood and microRNA (miRNA) expression in plasma, in a cohort of 20 females with Rett syndrome, 2 males with MECP2 duplication syndrome and 28 healthy controls, and correlated RNA expression with disease and clinical parameters. We have identified a set of potential biomarker panels for RTT diagnostic and disease stratification of patients with microcephaly and vasomotor deficits. Our study sets the basis for larger studies leading to the identification of specific miRNA signatures for early RTT detection, stratification, disease progression and segregation from other neurodevelopmental disorders. Nevertheless, these data will require verification and validation in further studies with larger sample size including a whole range of ages.

Molecular characterization of Spanish patients with MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

Enquiring beneath the surface: can a gene expression assay shed light into the heterogeneity among newborns with neonatal encephalopathy?

BACKGROUND: We aimed to assess whether a gene expression assay provided insights for understanding the heterogeneity among newborns affected by neonatal encephalopathy (NE). METHODS: Analysis by RT-qPCR of the mRNA expression of candidate genes in whole blood from controls (n = 34) and NE (n = 24) patients at <6, 12, 24, 48, 72 and 96 h of life, followed by determination of differences in gene expression between conditions and correlation with clinical variables. RESULTS: During the first 4 days of life, MMP9, PPARG, IL8, HSPA1A and TLR8 were more expressed and CCR5 less expressed in NE patients compared to controls. MMP9 and PPARG increased and CCR5 decreased in moderate/severe NE patients compared to mild. At 6-12 h of life, increased IL8 correlated with severe NE and death, decreased CCR5 correlated with chorioamnionitis and increased HSPA1A correlated with expanded multiorgan dysfunction, severe NE and female sex. CONCLUSIONS: MMP9, PPARG and CCR5 mRNA expression within first days of life correlates with the severity of NE. At 6-12 h, IL8 and HSPA1A are good reporters of clinical variables in NE patients. HSPA1A may have a role in the sexual dimorphism observed in NE. CCR5 is potentially involved in the link between severe NE and chorioamnionitis.

Comprehensive Analysis of GABAA-A1R Developmental Alterations in Rett Syndrome: Setting the Focus for Therapeutic Targets in the Time Frame of the Disease.

Rett syndrome, a serious neurodevelopmental disorder, has been associated with an altered expression of different synaptic-related proteins and aberrant glutamatergic and γ-aminobutyric acid (GABA)ergic neurotransmission. Despite its severity, it lacks a therapeutic option. Through this work we aimed to define the relationship between MeCP2 and GABAA.-A1 receptor expression, emphasizing the time dependence of such relationship. For this, we analyzed the expression of the ionotropic receptor subunit in different MeCP2 gene-dosage and developmental conditions, in cells lines, and in primary cultured neurons, as well as in different developmental stages of a Rett mouse model. Further, RNAseq and systems biology analysis was performed from post-mortem brain biopsies of Rett patients. We observed that the modulation of the MeCP2 expression in cellular models (both Neuro2a (N2A) cells and primary neuronal cultures) revealed a MeCP2 positive effect on the GABAA.-A1 receptor subunit expression, which did not occur in other proteins such as KCC2 (Potassium-chloride channel, member 5). In the Mecp2+/- mouse brain, both the KCC2 and GABA subunits expression were developmentally regulated, with a decreased expression during the pre-symptomatic stage, while the expression was variable in the adult symptomatic mice. Finally, the expression of the gamma-aminobutyric acid (GABA) receptor-related synaptic proteins from the postmortem brain biopsies of two Rett patients was evaluated, specifically revealing the GABA A1R subunit overexpression. The identification of the molecular changes along with the Rett syndrome prodromic stages strongly endorses the importance of time frame when addressing this disease, supporting the need for a neurotransmission-targeted early therapeutic intervention.

Discovery of Biomarker Panels for Neural Dysfunction in Inborn Errors of Amino Acid Metabolism.

Patients with inborn errors of amino acid metabolism frequently show neuropsychiatric symptoms despite accurate metabolic control. This study aimed to gain insight into the underlying mechanisms of neural dysfunction. Here we analyzed the expression of brain-derived neurotrophic factor (BDNF) and 10 genes required for correct brain functioning in plasma and blood of patients with Urea Cycle Disorders (UCD), Maple Syrup Urine Disease (MSUD) and controls. Receiver-operating characteristic (ROC) analysis was used to evaluate sensitivity and specificity of potential biomarkers. CACNA2D2 (α2δ2 subunit of voltage-gated calcium channels) and MECP2 (methyl-CpG binding protein 2) mRNA and protein showed an excellent neural function biomarker signature (AUC ≥ 0,925) for recognition of MSUD. THBS3 (thrombospondin 3) mRNA and AABA gave a very good biomarker signature (AUC 0,911) for executive-attention deficits. THBS3, LIN28A mRNA, and alanine showed a perfect biomarker signature (AUC 1) for behavioral and mood disorders. Finally, a panel of BDNF protein and at least two large neural AAs showed a perfect biomarker signature (AUC 1) for recognition of psychomotor delay, pointing to excessive protein restriction as central causative of psychomotor delay. To conclude, our study has identified promising biomarker panels for neural function evaluation, providing a base for future studies with larger samples.

Differential neuronal and glial behavior on flat and micro patterned chitosan films.

Chitosan is a biodegradable natural polysaccharide that has been widely studied for regenerative purposes in the central nervous system. In this study we assessed the in vitro glial and neuronal cells response to chitosan either flat or patterned with grooves in the micrometric range. Chitosan demonstrated to be a good substrate for the attachment and growth of both neurons and glial cells. Chitosan micropatterns promoted glial cell maturation, suggesting astroglial activation. Nevertheless, those mature/reactive glial cells were permissive for axonal growth. Axons aligned and organized along the patterned grooves and the size of the linear topographic patterns is also affecting neurite and cell response. Patterns with 10µm width induced fasciculation of axons, which can be useful for CNS tissue engineering substrates when precise orientation of the axonal outgrowth is desired.

Learning and memory disabilities in IUGR babies: Functional and molecular analysis in a rat model.

INTRODUCTION: 1Intrauterine growth restriction (IUGR) is the failure of the fetus to achieve its inherent growth potential, and it has frequently been associated with neurodevelopmental problems in childhood. Neurological disorders are mostly associated with IUGR babies with an abnormally high cephalization index (CI) and a brain sparing effect. However, a similar correlation has never been demonstrated in an animal model. The aim of this study was to determine the correlations between CI, functional deficits in learning and memory and alterations in synaptic proteins in a rat model of IUGR. METHODS: 2Utero-placental insufficiency was induced by meso-ovarian vessel cauterization (CMO) in pregnant rats at embryonic day 17 (E17). Learning performance in an aquatic learning test was evaluated 25 days after birth and during 10 days. Some synaptic proteins were analyzed (PSD95, Synaptophysin) by Western blot and immunohistochemistry. RESULTS: 3Placental insufficiency in CMO pups was associated with spatial memory deficits, which are correlated with a CI above the normal range. CMO pups presented altered levels of synaptic proteins PSD95 and synaptophysin in the hippocampus. CONCLUSIONS: 4The results of this study suggest that learning disabilities may be associated with altered development of excitatory neurotransmission and synaptic plasticity. Although interspecific differences in fetal response to placental insufficiency should be taken into account, the translation of these data to humans suggest that both IUGR babies and babies with a normal birth weight but with intrauterine Doppler alterations and abnormal CI should be closely followed to detect neurodevelopmental alterations during the postnatal period.

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1del/del mice, unmasking role in non-gluconeogenic tissues

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism (AU)

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PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

Neuronal Progenitor Maintenance Requires Lactate Metabolism and PEPCK-M-Directed Cataplerosis.

This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.

p38α function in osteoblasts influences adipose tissue homeostasis.

The skeleton acts as an endocrine organ that regulates energy metabolism and calcium and phosphorous homeostasis through the secretion of osteocalcin (Oc) and fibroblast growth factor 23 (FGF23). However, evidence suggests that osteoblasts secrete additional unknown factors that contribute to the endocrine function of bone. To search for these additional factors, we generated mice with a conditional osteoblast-specific deletion of p38α MAPK known to display profound defects in bone homeostasis. Herein, we show that impaired osteoblast function is associated with a strong decrease in body weight and adiposity (P < 0.01). The differences in adiposity were not associated with diminished caloric intake, but rather reflected 20% increased energy expenditure and the up-regulation of uncoupling protein-1 (Ucp1) in white adipose tissue (WAT) and brown adipose tissue (BAT) (P < 0.05). These alterations in lipid metabolism and energy expenditure were correlated with a decrease in the blood levels of neuropeptide Y (NPY) (40% lower) rather than changes in the serum levels of insulin, Oc, or FGF23. Among all Npy-expressing tissues, only bone and primary osteoblasts showed a decline in Npy expression (P < 0.01). Moreover, the intraperitoneal administration of recombinant NPY partially restored the WAT weight and adipocyte size of p38α-deficient mice (P < 0.05). Altogether, these results further suggest that, in addition to Oc, other bone-derived signals affect WAT and energy expenditure contributing to the regulation of energy metabolism.

An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA.

BACKGROUND: Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-µm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. NEW METHOD: Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. RESULTS: Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. COMPARISON WITH EXISTING METHODS: The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. CONCLUSIONS: The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications.

Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold.

Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-l/dl lactic acid (PLA70/30) nanofibers release L-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while L-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the in vivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation.

The effect of the composition of PLA films and lactate release on glial and neuronal maturation and the maintenance of the neuronal progenitor niche.

To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly L/DL lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. L-lactate in the medium recapitulated PLA70/30's maintenance of neuronal restricted progenitors but did not sustain bipotential or glial restricted progenitors in the cultures, as occurred when neural cells were grown on PLA70/30. Our results suggest that PLA70/30 may mimic some of the physical and biochemical characteristics of the NSC niche. Its mechanical and surface properties may act synergistically in the modulation of bipotential and glial restricted progenitor phenotypes, while it is L-lactate, either added to the medium or released by the film that drives the maintenance of neuronal restricted progenitor cell phenotypes.

Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA.

Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to de-differentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 µm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.

Appropriate Bmp7 levels are required for the differentiation of midline guidepost cells involved in corpus callosum formation.

Guidepost cells are essential structures for the establishment of major axonal tracts. How these structures are specified and acquire their axon guidance properties is still poorly understood. Here, we show that in mouse embryos appropriate levels of Bone Morphogenetic Protein 7 (Bmp7), a member of the TGF-ß superfamily of secreted proteins, are required for the correct development of the glial wedge, the indusium griseum, and the subcallosal sling, three groups of cells that act as guidepost cells for growing callosal axons. Bmp7 is expressed in the region occupied by these structures and its genetic inactivation in mouse embryos caused a marked reduction and disorganization of these cell populations. On the contrary, infusion of recombinant Bmp7 in the developing forebrain induced their premature differentiation. In both cases, changes were associated with the disruption of callosal axon growth and, in most animals fibers did not cross the midline forming typical Probst bundles. Addition of Bmp7 to cortical explants did not modify the extent of their outgrowth nor their directionality, when explants were exposed to a focalized source of the protein. Together, these results indicate that Bmp7 is indirectly required for corpus callosum formation by controlling the timely differentiation of its guidepost cells.

BDNF/MAPK/ERK-induced BMP7 expression in the developing cerebral cortex induces premature radial glia differentiation and impairs neuronal migration.

During development of the mammalian nervous system, a combination of genetic and environmental factors governs the sequential generation of neurons and glia and the initial establishment of the neural circuitry. Here, we demonstrate that brain-derived neurotrophic factor (BDNF), one of those local acting factors, induces Bone Morphogenetic Protein 7 (BMP7) expression in embryonic neurons by activating Mitogen-Activated Protein Kinase/Extracellular signal-Regulated Kinase signaling and by the negative regulation of p53/p73 function. We also show that intraventricular injection of BMP7 at midgestation induces the early differentiation of radial glia into glial precursors and astrocytes and the expression of mature glial markers such as the antiadhesive protein SC1. As a result of this precocious radial glia maturation, the laminar distribution of late-born pyramidal neurons is altered, most likely by the termination of radial glia ability to support neuronal migration and the early neuronal detachment from the glial rail. We propose a mechanism for BDNF regulation of BMP7 in which local activity-driven BDNF-induced BMP7 expression at the end of neurogenesis instructs competent precursors to generate astrocytes. Such a mechanism might ensure synchronic neuronal and glial maturation at the beginning of cortical activity.