RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS+IFNγ

Microglia, the main resident immune cells in the CNS, are thought to participate in the pathogenesis of various neurological disorders. LPS and LPS + IFNγ are stimuli that are widely used to activate microglia. However, the transcriptomic profiles of microglia treated with LPS and LPS + IFNγ have not been properly compared. Here, we treated murine primary microglial cultures with LPS or LPS + IFNγ for 6 hours and then performed RNA-Sequencing. Gene expression patterns induced by the treatments were obtained by WGCNA and 11 different expression profiles were found, showing differential responses to LPS and LPS + IFNγ in many genes. Interestingly, a subset of genes involved in Parkinson's, Alzheimer's and Huntington's disease were downregulated by both treatments. By DESeq analysis we found differentially upregulated and downregulated genes that confirmed LPS and LPS + IFNγ as inducers of microglial pro-inflammatory responses, but also highlighted their involvement in specific cell functions. In response to LPS, microglia tended to be more proliferative, pro-inflammatory and phagocytic; whereas LPS + IFNγ inhibited genes were involved in pain, cell division and, unexpectedly, production of some inflammatory mediators. In summary, this study provides a detailed description of the transcriptome of LPS- and LPS + IFNγ treated primary microglial cultures. It may be useful to determine whether these in vitro phenotypes resemble microglia in in vivo pathological conditions

Deficits in coordinated neuronal activity and network topology are striatal hallmarks in Huntington's disease.

Background. Network alterations underlying neurodegenerative diseases often precede symptoms and functional deficits. Thus, their early identification is central for improved prognosis. In Huntington's disease (HD), the cortico-striatal networks, involved in motor function processing, are the most compromised neural substrate. However, whether the network alterations are intrinsic of the striatum or the cortex is not fully understood. Results In order to identify early HD neural deficits, we characterized neuronal ensemble calcium activity and network topology of HD striatal and cortical cultures. We used large-scale calcium imaging combined with activity-based network inference analysis. We extracted collective activity events and inferred the topology of the neuronal network in cortical and striatal primary cultures from wild-type and R6/1 mouse model of HD. Striatal, but not cortical, HD networks displayed lower activity and a lessened ability to integrate information. GABAA receptor blockade in healthy and HD striatal cultures generated similar coordinated ensemble activity and network topology, highlighting that the excitatory component of striatal system is spared in HD. Conversely, NMDA receptor activation increased individual neuronal activity while coordinated activity became highly variable and undefined. Interestingly, by boosting NMDA activity, we rectified striatal HD network alterations. Conclusions. Overall, our integrative approach highlights striatal defective network integration capacity as a major contributor of basal ganglia dysfunction in HD and suggests that increased excitatory drive may serve as a potential intervention. In addition, our work provides a valuable tool to evaluate in vitro network recovery after treatment intervention in basal ganglia disorders

Pharmacogenetic Modulation of STEP improves motor and cognitive function in a mouse model of Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expansion of a CAG repeat in the huntingtin (htt) gene, which results in an aberrant form of the protein (mhtt). This leads to motor and cognitive deficits associated with corticostriatal and hippocampal alterations. The levels of STriatal-Enriched protein tyrosine Phosphatase (STEP), a neural-specific tyrosine phosphatase that opposes the development of synaptic strengthening, are decreased in the striatum of HD patients and also in R6/1 mice, thereby contributing to the resistance to excitotoxicity described in this HD mouse model. Here, we aimed to analyze whether STEP inactivation plays a role in the pathophysiology of HD by investigating its effect on motor and cognitive impairment in the R6/1 mouse model of HD. We found that genetic deletion of STEP delayed the onset of motor dysfunction and prevented the appearance of cognitive deficits in R6/1 mice. This phenotype was accompanied by an increase in pERK1/2 levels, a delay in the decrease of striatal DARPP-32 levels and a reduction in the size of mhtt aggregates, both in the striatum and CA1 hippocampal region. We also found that acute pharmacological inhibition of STEP with TC-2153 improved cognitive function in R6/1 mice. In conclusion, our results show that deletion of STEP has a beneficial effect on motor coordination and cognition in a mouse model of HD suggesting that STEP inhibition could be a good therapeutic strategy in HD patients

Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice

Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time

M2 Cortex-Dorsolateral striatum stimulation reverses motor symptoms and synaptic deficits in Huntington's Disease

Huntington's disease (HD) is a neurological disorder characterized by motor disturbances. HD pathology is most prominent in the striatum, the central hub of the basal ganglia. The cerebral cortex is the main striatal afferent, and progressive cortico-striatal disconnection characterizes HD. We mapped striatal network dysfunction in HD mice to ultimately modulate the activity of a specific cortico-striatal circuit to ameliorate motor symptoms and recover synaptic plasticity. Multimodal MRI in vivo indicates cortico-striatal and thalamo-striatal functional network deficits and reduced glutamate/glutamine ratio in the striatum of HD mice. Moreover, optogenetically-induced glutamate release from M2 cortex terminals in the dorsolateral striatum (DLS) was undetectable in HD mice and striatal neurons show blunted electrophysiological responses. Remarkably, repeated M2-DLS optogenetic stimulation normalized motor behavior in HD mice and evoked a sustained increase of synaptic plasticity. Overall, these results reveal that selective stimulation of the M2-DLS pathway can become an effective therapeutic strategy in HD

Myeloid C/EBPβ deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis

Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPβ show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPβ-expressing cell types is not solved. Since C/EBPβ-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPβ deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPβ deficiency. Methods Mice with myeloid C/EBPβ deficiency were generated by crossing LysMCre and C/EBPβfl/fl mice. Primary microglial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPβ deletion were analyzed in vivo in microglia isolated from the brains of C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPβfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. Results LysMCre-C/EBPβfl/fl mice showed an efficiency of C/EBPβ deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPβ deficiency. Transcriptomic analysis of C/EBPβ-deficient primary microglia revealed C/EBPβ-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPβ deletion. CNS expression of C/EBPβ was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPβfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. Conclusion This study provides new data that support a central role for C/EBPβ in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPβ inhibition in multiple sclerosis

Inflammation in multiple sclerosis induces a specific reactive astrocyte state driving non-cell-autonomous neuronal damage

An in‐depth understanding of the neurodegenerative component of multiple sclerosis (MS) is crucial for the design of therapeutic approaches that may stop disease progression. Astrocytes have emerged as key contributors to the pathogenesis of MS. 1 However, the mechanisms underlying the regulation of maladaptive astrocytic responses remain unknown. In this report, we show that a high inflammatory activity in MS patients at disease onset induces a specific reactive astrocyte state that triggers synaptopathy and contributes to neuronal damage in vitro and ex vivo suggesting potential mechanisms that may ultimately lead to neurodegeneration. To investigate whether astrocytes are essential contributors to neuronal damage in MS, we cultured purified astrocytes with cerebrospinal fluid (CSF) samples from MS patients with high inflammatory activity at disease onset (MS‐High, Table S1). Then, we examined the effect of astrocytic secretomes on neurons (Figure 1A). Astrocytes became reactive upon high inflammatory CSF exposure (Figure 1B) and induced morphological alterations typically observed in neurodegenerative disorders, such as a less complex dendritic tree due to decreased arborisation (Figure 1C, D). Moreover, these abnormalities were accompanied with synaptic plasticity impairment (Figure 1E, F). Considering that a high lesion load at disease onset has been associated with an increased risk of neurological disability development, 2 we assessed whether the non‐cell‐autonomous effect on neuronal plasticity could be influenced by the degree of inflammatory activity of MS patients (Figure 2A and Table S1). Interestingly, we observed a direct correlation between the degree of inflammatory exposure and the extent of both astrocyte‐mediated synaptopathy (Figure 2B, C) and dendrite arborisation impairment

Helios expression coordinates the development of a subset of striatopallidal medium spiny neurons

Here we unravel the mechanism of action of Helios (He) during the development of striatal medium spiny neurons (MSNs). He regulates the second wave of striatal neurogenesis involved in the generation of striatopallidal neurons that express dopamine 2 receptor (D2R) and enkephalin (ENK). To exert this effect He is expressed in neural progenitor cells (NPCs) retaining them into the G1/G0 phase of the cell cycle. Thus, the lack of He produces an increase of S-phase entry and S-phase length of NPCs which in turn impairs striatal neurogenesis and produces an accumulation of the number of cycling NPCs in the germinal zone (GZ) that end up dying at postnatal stages. Therefore, He-/- mice show a reduction in the number of Dorso-Medial Striatal MSNs in the adulthood that produces deficits in motor skills acquisition. In addition, overexpression of He in NPCs induce DARPP32 phenotype when transplanted in mouse striatum.Present findings demonstrate that He is involved in the correct development of a subset of striatopallidal MSNs and reveal new cellular mechanisms for neuronal development

Piaget en la educación : Debate en torno de sus aportaciones

Tabla de contenidos: La teoría genética y los procesos de construcción del conocimiento en el aula / César Coll. Los problemas de una teoría del aprendizaje: una discusión crítica de la tradición psicogenética / José Antonio Castorina. Una aproximación al análisis de la influencia de la obra piagetiana en la educación / Frida Díaz Barriga Areco, Gerardo Hernández, Benilde García, Irene Muriá. Piaget: aportes para la educación y para la didáctica / Angel Díaz Barriga. Cambio de base epistemológica en la enseñanza de las ciencias: actividades de experimentación / Anna Maria Pessoa de Carvalho. La enseñanza de la matemática: un enfoque constructivista / Luis Moreno Armella. La enseñanza de la física y de las matemáticas desde la epistemología piagetiana / Carlos Eduardo Vasco.Fil: Castorina, José Antonio. Universidad Nacional de La Plata. Facultad de Humanidades y Ciencias de la Educación; Argentina

Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery