Trem2 expression in microglia is required to maintain normal neuronal bioenergetics during development.

Triggering receptor expressed on myeloid cells 2 (Trem2) is a myeloid cell-specific gene expressed in brain microglia, with variants that are associated with neurodegenerative diseases, including Alzheimer's disease. Trem2 is essential for microglia-mediated synaptic refinement, but whether Trem2 contributes to shaping neuronal development remains unclear. Here, we demonstrate that Trem2 plays a key role in controlling the bioenergetic profile of pyramidal neurons during development. In the absence of Trem2, developing neurons in the hippocampal cornus ammonis (CA)1 but not in CA3 subfield displayed compromised energetic metabolism, accompanied by reduced mitochondrial mass and abnormal organelle ultrastructure. This was paralleled by the transcriptional rearrangement of hippocampal pyramidal neurons at birth, with a pervasive alteration of metabolic, oxidative phosphorylation, and mitochondrial gene signatures, accompanied by a delay in the maturation of CA1 neurons. Our results unveil a role of Trem2 in controlling neuronal development by regulating the metabolic fitness of neurons in a region-specific manner.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring.

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Identification of a choroid plexus vascular barrier closing during intestinal inflammation.

Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/ß-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel disease­related mental symptoms may thus be the consequence of a deregulated gut­brain vascular axis.

Design of transfections: Implementation of design of experiments for cell transfection fine tuning.

Transfection is the process by which nucleic acids are introduced into eukaryotic cells. This is fundamental in basic research for studying gene function and modulation of gene expression as well as for many bioprocesses in the manufacturing of clinical-grade recombinant biologics from cells. Transfection efficiency is a critical parameter to increase biologics' productivity; the right protocol has to be identified to ensure high transfection efficiency and therefore high product yield. Design of experiments (DoE) is a mathematical method that has become a key tool in bioprocess development. Based on the DoE method, we developed an operational flow that we called "Design of Transfections" (DoT) for specific transfection modeling and identification of the optimal transfection conditions. As a proof of principle, we applied the DoT workflow to optimize a cell transfection chemical protocol for neural progenitors, using polyethyleneimine (PEI). We simultaneously varied key influencing factors, namely concentration and type of PEI, DNA concentration, and cell density. The transfection efficiency was measured by fluorescence imaging followed by automatic counting of the green fluorescent transfected cells. Taking advantage of the DoT workflow, we developed a new simple, efficient, and economically advantageous PEI transfection protocol through which we were able to obtain a transfection efficiency of 34%.

The Transcription Regulator Patz1 Is Essential for Neural Stem Cell Maintenance and Proliferation.

Proper regulation of neurogenesis, the process by which new neurons are generated from neural stem and progenitor cells (NS/PCs), is essential for embryonic brain development and adult brain function. The transcription regulator Patz1 is ubiquitously expressed in early mouse embryos and has a key role in embryonic stem cell maintenance. At later stages, the detection of Patz1 expression mainly in the developing brain suggests a specific involvement of Patz1 in neurogenesis. To address this point, we first got insights in Patz1 expression profile in different brain territories at both embryonic and postnatal stages, evidencing a general decreasing trend with respect to time. Then, we performed in vivo and ex vivo analysis of Patz1-knockout mice, focusing on the ventricular and subventricular zone, where we confirmed Patz1 enrichment through the analysis of public RNA-seq datasets. Both embryos and adults showed a significant reduction in the number of Patz1-null NS/PCs, as well as of their self-renewal capability, compared to controls. Consistently, molecular analysis revealed the downregulation of stemness markers in NS/PCs derived from Patz1-null mice. Overall, these data demonstrate the requirement of Patz1 for NS/PC maintenance and proliferation, suggesting new roles for this key transcription factor specifically in brain development and plasticity, with possible implications for neurodegenerative disorders and glial brain tumors.

Early maternal care restores LINE-1 methylation and enhances neurodevelopment in preterm infants.

BACKGROUND: Preterm birth affects almost 9-11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice. METHODS: Very preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants' neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages. RESULTS: Here we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting. CONCLUSIONS: Our results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants' prematurity-related neurodevelopmental outcomes. TRIAL REGISTRATION: ClinicalTrial.gov ( NCT02983513 ). Registered on 6 December 2016, retrospectively registered.

Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue.

Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Decoding neuronal diversity in the developing cerebral cortex: from single cells to functional networks.

The neocortex is by far one of the most complex regions of the mammalian brain, characterized by an extraordinary diversity of neuronal and non-neuronal cell types, whose coordinated development and function guarantee the execution of high-order cognitive, sensory, and motor behaviours. Decoding its heterogeneity and understanding the molecular strategies upon which the cerebral cortex is built during development have been at the core of neuroscientists' work for decades. Here, we will focus on the current classification of neuronal types (both excitatory and inhibitory) of the neocortex in light of the insights provided by recent single-cell omic technologies, which have offered - with unprecedented resolution - an extended framework to interpret cortical diversity and its developmental origin. We will cover the impact of neuronal subtype identity on generating specific neuronal networks (neuron-to-neuron interaction), as well as their effect on the development of the non-neuronal populations in the cerebral cortex.

Cripto haploinsufficiency affects in vivo colon tumor development.

Colorectal cancer is one of the most common and aggressive cancers arising from alterations in various signaling pathways, such as the WNT, RAS-MAPK, PI3K and transforming growth factor-ß (TGF-ß) pathways. Cripto (also called Teratocarcinoma-derived growth factor), the original member of the vertebrate EGF-CFC family, plays a key role in all of these pathways and is deeply involved in early embryo development and cancer progression. The role of Cripto in colon and breast cancer, in particular, has been investigated, as it is still not clearly understood. In this article, we provide the first in vivo functional evidence of a role of Cripto in colon cancer development. We analyzed the effect of Cripto haploinsufficiency on colon tumor formation by treating Cripto heterozygous mice with the colonotropic carcinogen azoxymethane (AOM). Of note, in our model system, Cripto haploinsufficiency increased tumorigenesis. Moreover, we revealed a correlation between the differential AOM response found in wt and Cripto⁺/⁻ mice and the expression levels of glucose regulated protein-78 (Grp78), a heat shock protein required for Cripto signaling pathways. We hypothesize that the balance between Cripto and Grp78 expression levels might be crucial in cancer development and may account for the increased tumorigenesis in Cripto heterozygous mice. In summary, our results highlight the heterogeneous effect of Cripto on tumorigenesis and the consequent high level of complexity in the Cripto regulatory pathway, whose imbalance causes tumors.