From Entry to the Nucleus: How Retroviruses Commute.

Once inside host cells, retroviruses generate a double-stranded DNA copy of their RNA genomes via reverse transcription inside a viral core, and this viral DNA is subsequently integrated into the genome of the host cell. Before integration can occur, the core must cross the cell cortex, be transported through the cytoplasm, and enter the nucleus. Retroviruses have evolved different mechanisms to accomplish this journey. This review examines the various mechanisms retroviruses, especially HIV-1, have evolved to commute throughout the cell. Retroviruses cross the cell cortex while modulating actin dynamics and use microtubules as roads while connecting with microtubule-associated proteins and motors to reach the nucleus. Although a clearer picture exists for HIV-1 compared with other retroviruses, there is still much to learn about how retroviruses accomplish their commute.

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1 G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2+ transients and reactive oxygen species (i.e., H 2 O 2 ). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior.

BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5' and 3'LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5'UTR sense is unable to initiate translation, whereas the antisense 5'UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5'UTR IRES activity was tested using bicistronic reporters. The antisense 5'UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon.

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

KMT2C knockout generates ASD-like behaviors in mice.

Neurodevelopmental disorders have been associated with genetic mutations that affect cellular function, including chromatin regulation and epigenetic modifications. Recent studies in humans have identified mutations in KMT2C, an enzyme responsible for modifying histone tails and depositing H3K4me1 and H3K4me3, as being associated with Kleefstra syndrome 2 and autism spectrum disorder (ASD). However, the precise role of KMT2C mutations in brain disorders remains poorly understood. Here we employed CRISPR/Cas9 gene editing to analyze the effects of KMT2C brain specific knockout on animal behavior. Knocking out KMT2C expression in cortical neurons and the mouse brain resulted in decreased KMT2C levels. Importantly, KMT2C brain specific knockout animals exhibited repetitive behaviors, social deficits, and intellectual disability resembling ASD. Our findings shed light on the involvement of KMT2C in neurodevelopmental processes and establish a valuable model for elucidating the cellular and molecular mechanisms underlying KMT2C mutations and their relationship to Kleefstra syndrome 2 and ASD.

Antiviral Activity of an Endogenous Parvoviral Element.

Endogenous viral elements (EVEs) are genomic DNA sequences derived from viruses. Some EVEs have open reading frames (ORFs) that can express proteins with physiological roles in their host. Furthermore, some EVEs exhibit a protective role against exogenous viral infection in their host. Endogenous parvoviral elements (EPVs) are highly represented in mammalian genomes, and although some of them contain ORFs, their function is unknown. We have shown that the locus EPV-Dependo.43-ODegus, an EPV with an intact ORF, is transcribed in Octodon degus (degu). Here we examine the antiviral activity of the protein encoded in this EPV, named DeRep. DeRep was produced in bacteria and used to generate antibodies that recognize DeRep in western blots of degu tissue. To test if DeRep could protect against exogenous parvovirus, we challenged cells with the minute virus of mice (MVM), a model autonomous parvovirus. We observed that MVM protein expression, DNA damage induced by replication, viral DNA, and cytopathic effects are reduced when DeRep is expressed in cells. The results of this study demonstrate that DeRep is expressed in degu and can inhibit parvovirus replication. This is the first time that an EPV has been shown to have antiviral activity against an exogenous virus.

Deletion of VPS50 protein in mice brain impairs synaptic function and behavior.

VPS50, is an accessory protein, involved in the synaptic and dense core vesicle acidification and its alterations produce behavioral changes in C.elegans. Here, we produce the mosaic knock out (mKO) of VPS50 using CRISPR/Cas9 system in both cortical cultured neurons and whole animals to evaluate the effect of VPS50 in regulating mammalian brain function and behavior. While mKO of VPS50 does not change the number of synaptic vesicles, it produces a mislocalization of the V-ATPase pump that likely impact in vesicle acidification and vesicle content to impair synaptic and neuronal activity in cultured neurons. In mice, mKO of VPS50 in the hippocampus, alter synaptic transmission and plasticity, and generated robust cognitive impairments associate to memory formation. We propose that VPS50 is an accessory protein that aids the correct recruitment of the V-ATPase pump to synaptic vesicles, thus having a crucial role controlling synaptic vesicle acidification and hence synaptic transmission.

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Using the ASD and anxiety mouse model Flailer, that contains a duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700bp genomic duplication in vitro and in vivo . Importantly, DN-CRISPRs have not been used to remove more gene regions <100bp successfully and with high efficiency. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene edition. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescues some of the mutant behaviors, while intracerebroventricular delivery, completely recovers Flailer animal phenotype associated to anxiety and ASD. Our results demonstrate the potential of DN-CRISPR to efficiently (>60% editing in vivo) remove large genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

Training on Emotional Intelligence for Caregivers of Patients with Acquired Brain Injury and Cognitive Impairment: A Quasi-Experimental Study.

BACKGROUND: Cognitive-behavioral alterations can occur after an acquired brain injury (ABI). OBJECTIVES: To develop and evaluate a synchronous online training program on emotional intelligence (EI) for the caregivers of adult patients with cognitive-behavioral impairment due to ABI. METHODS: Quasi-experimental study. Ten caregivers attended a one-month virtual synchronous course about EI. The emotional status of the caregivers was registered one-month-previous and one-month-post program using comparative measures: The Trait Meta-Mood Scale (TMMS-24), the Positive and Negative Affect Schedule (PANAS), Caregiver Burden Interview, the 10-item Connor-Davidson Resilience Scale, and the Emotional Health Survey. RESULTS: After the training course, the favorable changes related to emotional affect measured with the PANAS questionnaire were found; both positive (increase; Mdn = 39.5; effect size -12.79; adjusted variance 95.75) and negative (decrease; Mdn = 14.5; effect size 0.73; adjusted variance 95.50) presented a statistical significance of p < 0.05. The TMMS-24 post-test showed that 90% of the caregivers reported an adequate or excellent emotional repair (p < 0.05; effect size -0.68; adjusted variance 94.75). No other significant differences were found. CONCLUSIONS: After this training in EI, the caregivers had a more positive mood and improved aspects of their emotional intelligence, such as emotional regulation. More studies need to be conducted.

Reactive Extrusion as a Pretreatment in Cassava (Manihot esculenta Crantz) and Pea (Pisum sativum L.) Starches to Improve Spinnability Properties for Obtaining Fibers.

Starch is a biocompatible and economical biopolymer in which interest has been shown in obtaining electrospun fibers. This research reports that cassava (CEX) and pea (PEX) starches pretreated by means of reactive extrusion (REX) improved the starches rheological properties and the availability of amylose to obtain fibers. Solutions of CEX and PEX (30-36% w/v) in 38% v/v formic acid were prepared and the rheological properties and electrospinability were studied. The rheological values indicated that to obtain continuous fibers without beads, the entanglement concentration (Ce) must be 1.20 and 1.25 times the concentration of CEX and PEX, respectively. In CEX, a higher amylose content and lower viscosity were obtained than in PEX, which resulted in a greater range of concentrations (32-36% w/v) to obtain continuous fibers without beads with average diameters ranging from 316 ± 65 nm to 394 ± 102 nm. In PEX, continuous fibers without beads were obtained only at 34% w/v with an average diameter of 170 ± 49 nm. This study showed that starches (20-35% amylose) pretreated through REX exhibited electrospinning properties to obtain fibers, opening the opportunity to expand their use in food, environmental, biosensor, and biomedical applications, as vehicles for the administration of bioactive compounds.

Effect of Microwave Irradiation on Acid Hydrolysis of Faba Bean Starch: Physicochemical Changes of the Starch Granules.

Starch is the most abundant carbohydrate in legumes (22-45 g/100 g), with distinctive properties such as high amylose and resistant starch content, longer branch chains of amylopectin, and a C-type pattern arrangement in the granules. The present study concentrated on the investigation of hydrolyzed faba bean starch using acid, assisted by microwave energy, to obtain a possible food-grade coating material. For evaluation, the physicochemical, morphological, pasting, and structural properties were analyzed. Hydrolyzed starches developed by microwave energy in an acid medium had low viscosity, high solubility indexes, diverse amylose contents, resistant starch, and desirable thermal and structural properties to be used as a coating material. The severe conditions (moisture, 40%; pure hydrochloric acid, 4 mL/100 mL; time, 60 s; and power level, 6) of microwave-treated starches resulted in low viscosity values, high amylose content and high solubility, as well as high absorption indexes, and reducing sugars. These hydrolyzed starches have the potential to produce matrices with thermo-protectants to formulate prebiotic/probiotic (symbiotic) combinations and amylose-based inclusion complexes for functional compound delivery. This emergent technology, a dry hydrolysis route, uses much less energy consumption in a shorter reaction time and without effluents to the environment compared to conventional hydrolysis.

Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons.

Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.

Emotional intelligence abilities of caregivers of patients with acquired brain injury and cognitive impairment: are they related to overload or resilience?

Introduction: Acquired brain injury entails stressful situations of emotional complexity. Objective: To evaluate possible relationship among emotional intelligence, emotional status, resilience, and burden sensation of caregivers of patients with cognitive-behavioral impairment due to acquired brain injury in isolation circumstances because of COVID-19 pandemic. Material and methods: An observational descriptive cross-sectional study of prevalence was designed with a sample of 17 caregivers of patients with cognitive-behavioral impairment due to acquired brain injury. Main outcome measures: Caregiver Burden Interview, the 10-item Connor-Davidson Resilience Scale, Emotional Health, the Trait Meta-Mood Scale and the Positive and Negative Affect Schedule. Results: The median age of the 17 caregivers was 47.5 years, 71% of women had a median care-time of 3.5 years, and 65% of the total sample were spouses of the patients. 70% of the patients were affected by stroke (hemorrhagic or ischemic cause). 59% of the caregivers presented a low level of emotional attention and emotional clarity, and 47 %, low emotional repair ability. 82% of them did not report overload, 53% showed low resilience level. Emotional intelligence showed strong correlation with resilience and mental health of caregivers. All of them were positively correlated with daily positive emotions and negatively correlated with negative emotions and overload. Conclusions: Emotional intelligence, resilience, and mental health of caregivers are strongly correlated. All of them increase positive emotions and reduce negative emotions and overload.

Introducción: el daño cerebral adquirido crea situaciones estresantes y de gran complejidad emocional. Objetivo: evaluar relación entre inteligencia emocional, resiliencia, estado emocional y sobrecarga de los cuidadores de pacientes con daño cerebral adquirido y afectación cognitivo-conductual en circunstancias de aislamiento por pandemia COVID-19. Materiales y métodos: estudio observacional descriptivo transversal de prevalencia. Participantes: 17 cuidadores de pacientes con daño cerebral adquirido y afectación cognitivo- conductual. Principales medidas: Cuestionario sobre Carga del Cuidador, Medida de Resiliencia Connor- Davidson, Cuestionario de Salud Emocional, Escala Trait Meta-Mood y Escala de Afecto Positivo y Negativo. Resultados: mediana de edad 47,5 años, 71% mujeres, mediana de tiempo siendo cuidador 3,5 años y 65% de la muestra eran cónyuges. El 70% de los pacientes habían sufrido un ictus y el principal deterioro cognitivo de ellos, referido por familiares, fue el déficit de memoria. El 59% de los cuidadores presentó bajo nivel de atención emocional y claridad emocional, y el 47%, baja capacidad de reparación emocional. El 82% no informó sobrecarga, el 53% mostró un bajo nivel de resiliencia y un afecto positivo en el mes previo ligeramente más alto que el afecto negativo. La inteligencia emocional mostró una fuerte correlación con la resiliencia y la salud mental. Además, estas tres variables correlacionaron positivamente con las emociones positivas y negativamente con las negativas y la sobrecarga. Conclusiones: Inteligencia emocional, resiliencia y salud mental están fuertemente correlacionadas. Las tres aumentan las emociones positivas y reducen las negativas y la sobrecarga. La sobrecarga asocia peor resiliencia, reparación emocional y salud mental.

Dynein Light-Chain Dynlrb2 Is Essential for Murine Leukemia Virus Traffic and Nuclear Entry.

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

SARS-CoV-2 infection in asymptomatic healthcare workers at a clinic in Chile.

Asymptomatic SARS-CoV-2 infection of healthcare workers (HCWs) has been reported as a key player in the nosocomial spreading of COVID-19. Early detection of infected HCWs can prevent spreading of the virus in hospitals among HCWs and patients. We conducted a cross-sectional study to determine the asymptomatic infection of HCWs in a private clinic in the city of Santiago, Chile. Our study was conducted during a period of 5 weeks at the peak of transmission of SARS-CoV-2 in Chile. Nasopharyngeal samples were obtained from 413 HCWs and tested for the presence of SARS-CoV-2 using RT-qPCR. We found that a 3.14% of HCWs were positive for the presence of SARS-CoV-2 (14/413). Out of these, 7/14 were completely asymptomatic and did not develop symptoms within 3 weeks of testing. Sequencing of viral genomes showed the predominance of the GR clade; however, sequence comparison demonstrated numerous genetic differences among them suggesting community infection as the main focus of transmission among HCWs. Our study demonstrates that the protocols applied to protect HCWs and patients have been effective as no infection clusters due to asymptomatic carriers were found in the clinic. Together, these data suggest that infection with SARS-CoV-2 among HCWs of this health center is not nosocomial.

Epigenetic silencing of the osteoblast-lineage gene program during hippocampal maturation.

Accumulating evidence indicates that epigenetic control of gene expression plays a significant role during cell lineage commitment and subsequent cell fate maintenance. Here, we assess epigenetic mechanisms operating in the rat brain that mediate silencing of genes that are expressed during early and late stages of osteogenesis. We report that repression of the osteoblast master regulator Sp7 in embryonic (E18) hippocampus is mainly mediated through the Polycomb complex PRC2 and its enzymatic product H3K27me3. During early postnatal (P10), juvenile (P30), and adult (P90) hippocampal stages, the repressive H3K27me3 mark is progressively replaced by nucleosome enrichment and increased CpG DNA methylation at the Sp7 gene promoter. In contrast, silencing of the late bone phenotypic Bglap gene in the hippocampus is PRC2-independent and accompanied by strong CpG methylation from E18 through postnatal and adult stages. Forced ectopic expression of the primary master regulator of osteogenesis Runx2 in embryonic hippocampal neurons activates the expression of its downstream target Sp7 gene. Moreover, transcriptomic analyses show that several genes associated with the mesenchymal-osteogenic lineages are transcriptionally activated in these hippocampal cells that express Runx2 and Sp7. This effect is accompanied by a loss in neuronal properties, including a significant reduction in secondary processes at the dendritic arbor and reduced expression of critical postsynaptic genes like PSD95. Together, our results reveal a developmental progression in epigenetic control mechanisms that repress the expression of the osteogenic program in hippocampal neurons at embryonic, postnatal, and adult stages.

Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124.

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3'-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.