Aberrant Somatosensory Processing and Connectivity in Mice Lacking Engrailed-2.

Overreactivity and defensive behaviors in response to tactile stimuli are common symptoms in autism spectrum disorder (ASD) patients. Similarly, somatosensory hypersensitivity has also been described in mice lacking ASD-associated genes such as Fmr1 (fragile X mental retardation protein 1). Fmr1 knock-out mice also show reduced functional connectivity between sensory cortical areas, which may represent an endogenous biomarker for their hypersensitivity. Here, we measured whole-brain functional connectivity in Engrailed-2 knock-out (En2-/-) adult mice, which show a lower expression of Fmr1 and anatomical defects common to Fmr1 knock-outs. MRI-based resting-state functional connectivity in adult En2-/- mice revealed significantly reduced synchronization in somatosensory-auditory/associative cortices and dorsal thalamus, suggesting the presence of aberrant somatosensory processing in these mutants. Accordingly, when tested in the whisker nuisance test, En2-/- but not WT mice of both sexes showed fear behavior in response to repeated whisker stimulation. En2-/- mice undergoing this test exhibited decreased c-Fos-positive neurons (a marker of neuronal activity) in layer IV of the primary somatosensory cortex and increased immunoreactive cells in the basolateral amygdala compared with WT littermates. Conversely, when tested in a sensory maze, En2-/- and WT mice spent a comparable time in whisker-guided exploration, indicating that whisker-mediated behaviors are otherwise preserved in En2 mutants. Therefore, fearful responses to somatosensory stimuli in En2-/- mice are accompanied by reduced basal connectivity of sensory regions, reduced activation of somatosensory cortex, and increased activation of the basolateral amygdala, suggesting that impaired somatosensory processing is a common feature in mice lacking ASD-related genes.SIGNIFICANCE STATEMENT Overreactivity to tactile stimuli is a common symptom in autism spectrum disorder (ASD) patients. Recent studies performed in mice bearing ASD-related mutations confirmed these findings. Here, we evaluated the behavioral response to whisker stimulation in mice lacking the ASD-related gene Engrailed-2 (En2-/- mice). Compared with WT controls, En2-/- mice showed reduced functional connectivity in the somatosensory cortex, which was paralleled by fear behavior, reduced activation of somatosensory cortex, and increased activation of the basolateral amygdala in response to repeated whisker stimulation. These results suggest that impaired somatosensory signal processing is a common feature in mice harboring ASD-related mutations.

Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance.

Autism spectrum disorders (ASD) and epilepsy are common neurological diseases of childhood, with an estimated incidence of approximately 0.5-1% of the worldwide population. Several genetic, neuroimaging and neuropathological studies clearly showed that both ASD and epilepsy have developmental origins and a substantial degree of heritability. Most importantly, ASD and epilepsy frequently coexist in the same individual, suggesting a common neurodevelopmental basis for these disorders. Genome-wide association studies recently allowed for the identification of a substantial number of genes involved in ASD and epilepsy, some of which are mutated in syndromes presenting both ASD and epilepsy clinical features. At the cellular level, both preclinical and clinical studies indicate that the different genetic causes of ASD and epilepsy may converge to perturb the excitation/inhibition (E/I) balance, due to the dysfunction of excitatory and inhibitory circuits in various brain regions. Metabolic and immune dysfunctions, as well as environmental causes also contribute to ASD pathogenesis. Thus, an E/I imbalance resulting from neurodevelopmental deficits of multiple origins might represent a common pathogenic mechanism for both diseases. Here, we will review the most significant studies supporting these hypotheses. A deeper understanding of the molecular and cellular determinants of autism-epilepsy comorbidity will pave the way to the development of novel therapeutic strategies.

Synthesis of 2,6-Diamino-Substituted Purine Derivatives and Evaluation of Cell Cycle Arrest in Breast and Colorectal Cancer Cells.

Reversine is a potent antitumor 2,6-diamino-substituted purine acting as an Aurora kinases inhibitor and interfering with cancer cell cycle progression. In this study we describe three reversine-related molecules, designed by docking calculation, that present structural modifications in the diamino units at positions 2 and 6. We investigated the conformations of the most stable prototropic tautomers of one of these molecules, the N6-cyclohexyl-N6-methyl-N2-phenyl-7H-purine-2,6-diamine (3), by Density Functional Theory (DFT) calculation in the gas phase, water and chloroform, the last solvent considered to give insights into the detection of broad signals in NMR analysis. In all cases the HN(9) tautomer resulted more stable than the HN(7) form, but the most stable conformations changed in different solvents. Molecules 1⁻3 were evaluated on MCF-7 breast and HCT116 colorectal cancer cell lines showing that, while being less cytotoxic than reversine, they still caused cell cycle arrest in G2/M phase and polyploidy. Unlike reversine, which produced a pronounced cell cycle arrest in G2/M phase in all the cell lines used, similar concentrations of 1⁻3 were effective only in cells where p53 was deleted or down-regulated. Therefore, our findings support a potential selective role of these structurally simplified, reversine-related molecules in p53-defective cancer cells.

Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor ß, and Sonic Hedgehog Signaling.

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)ß signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFß and SHH signaling.

Hippocampal dysregulation of neurofibromin-dependent pathways is associated with impaired spatial learning in engrailed 2 knock-out mice.

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

The positional identity of mouse ES cell-generated neurons is affected by BMP signaling.

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior-posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.

Sex-biasing influence of autism-associated Ube3a gene overdosage at connectomic, behavioral, and transcriptomic levels.

Genomic mechanisms enhancing risk in males may contribute to sex bias in autism. The ubiquitin protein ligase E3A gene (Ube3a) affects cellular homeostasis via control of protein turnover and by acting as transcriptional coactivator with steroid hormone receptors. Overdosage of Ube3a via duplication or triplication of chromosomal region 15q11-13 causes 1 to 2% of autistic cases. Here, we test the hypothesis that increased dosage of Ube3a may influence autism-relevant phenotypes in a sex-biased manner. We show that mice with extra copies of Ube3a exhibit sex-biasing effects on brain connectomics and autism-relevant behaviors. These effects are associated with transcriptional dysregulation of autism-associated genes, as well as genes differentially expressed in 15q duplication and in autistic people. Increased Ube3a dosage also affects expression of genes on the X chromosome, genes influenced by sex steroid hormone, and genes sex-differentially regulated by transcription factors. These results suggest that Ube3a overdosage can contribute to sex bias in neurodevelopmental conditions via influence on sex-differential mechanisms.

Exon Skipping Through Chimeric Antisense U1 snRNAs to Correct Retinitis Pigmentosa GTPase-Regulator (RPGR) Splice Defect.

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator (RPGR) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming at restoring impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1_asRNAs) and tested their efficacy in vitro, upon transient cotransfection with RPGR minigene reporter systems in HEK-293T, 661W, and PC-12 cell lines. We thus identified three chimeric U1_asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5'antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T and 661W cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1_snRNA exon skipping-based approach to correct splicing defects in RPGR.

Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson's disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.

Noggin elicits retinal fate in Xenopus animal cap embryonic stem cells.

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse-transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c-fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.

Altered Expression of GABAergic Markers in the Forebrain of Young and Adult Engrailed-2 Knockout Mice.

Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.

AAV-miR-204 Protects from Retinal Degeneration by Attenuation of Microglia Activation and Photoreceptor Cell Death.

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

Seizures increase importin-beta1 expression in NG2+ cells in the rat hippocampus.

Importins, also called karyopherins, belong to a large family of proteins involved in cytoplasm-to-nucleus transport. Transport machinery generally involves a complex formed by two different importin subtypes (alpha and beta). Both alpha and beta importins are expressed in the brain, and their expression and localization is regulated by physiological neuronal activity. Little is known about regulation of importin expression in brain pathological conditions. Here we studied the expression of importin beta1 (imp beta 1) in the rat hippocampus after acute and chronic seizures induced by the glutamate agonist kainic acid (KA). The overall content of imp beta 1 mRNA and protein did not change after acute KA seizures. However, acute KA seizures rapidly induced the translocation of imp beta 1 protein from the cytoplasm to the nucleus in pyramidal CA1 neurons. KA-induced imp beta 1 translocation was prevented by the NMDA (N-methyl-D-aspartic acid) receptor blocker MK-801. After chronic seizures, the overall levels of imp beta 1 mRNA and protein did not change in the whole hippocampus. Immunohistochemistry revealed a massive loss of imp beta 1-positive neurons in pyramidal layers (that degenerated after KA), whereas an increased number of imp beta 1-positive cells was detected in the stratum radiatum of rats with chronic seizures compared with control animals. Double-labeling experiments identified these cells as glial cells expressing the chondroitin sulfate proteoglycan NG2 (neuron/glial antigen 2), a glial subtype recently shown to regulate hippocampal neuron excitability. These data show a differential regulation of imp beta 1 expression after acute and chronic seizure activity in the rat hippocampus.

Retinal defects in mice lacking the autism-associated gene Engrailed-2.

Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD) patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2-/-) mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2+/+ controls, En2-/- adult retinas showed a significant decrease in the number of calbindin+ horizontal cells, and a significant increase in calbindin+ amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2-/- retina, as shown by Brn3a+ cell counts. In addition, En2-/- adult mice showed a significant reduction of photoreceptor (rhodopsin) and bipolar cell (Pcp2, PKCα) markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2-/- mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model.

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors.

Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.

An Eye on the Wnt Inhibitory Factor Wif1.

The coordinated interplay between extrinsic activating and repressing cell signaling molecules is pivotal for embryonic development and subsequent tissue homeostasis. This is well exemplified by studies on the evolutionarily conserved Wnt signaling pathways. Tight temporal and spatial regulation of Wnt signaling activity is required throughout lifetime, from maternal stages before gastrulation until and throughout adulthood. Outside cells, the action of numerous Wnt ligands is counteracted and fine-tuned by only a handful of well characterized secreted inhibitors, such as for instance Dickkopf, secreted Frizzled Related Proteins and Cerberus. Here, we give an overview of our current understanding of another secreted Wnt signaling antagonist, the Wnt inhibitory factor Wif1. Wif1 can directly interact with various Wnt ligands and inhibits their binding to membrane bound receptors. Epigenetic promoter methylation of Wif1, leading to silencing of its transcription and concomitant up-regulation of Wnt signaling, is a common feature during cancer progression. Furthermore, an increasing number of reports describe Wif1 involvement in regulating processes during embryonic development, which so far has not received as much attention. We will summarize our knowledge on Wif1 function and its mode of action with a particular focus on the zebrafish (Danio rerio). In addition, we highlight the potential of Wif1 research to understand and possibly influence mechanisms underlying eye diseases and regeneration.

Impaired Neuronal Differentiation of Neural Stem Cells Lacking the Engrailed-2 Gene.

The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2hd/hd, referred to as En2-/-) show molecular, anatomical and behavioral "ASD-like" features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2-/- postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2+/+ and En2-/- mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2+/+ NSCs, En2-/- NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2-/- NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2-/- postnatal forebrain.

Expression of cytosolic branched chain aminotransferase (BCATc) mRNA in the developing mouse brain.

Branched-chain aminotransferase (BCAT) catalyzes the transamination of essential branched-chain amino acids (BCAAs: leucine, isoleucine and valine) with alpha-ketoglutarate. Through this reaction, BCAAs provide nitrogen for the synthesis of glutamate, the predominant excitatory neurotransmitter. Two BCAT isoforms have been identified: one cytosolic (BCATc) and one mitochondrial (BCATm). In adult rodents, BCATc is expressed in a wide variety of structures of the central nervous system (CNS), in neurons. So far, no data were available about the expression of BCATc in the developing CNS. Here, we analyse the expression profile of BCATc mRNA in the mouse brain from embryonic day 12.5 to adult age. BCATc mRNA gradually appears in different brain regions starting from early stages of neural development, and is maintained until adulthood. BCATc mRNA is predominantly present in the cerebral cortex, hippocampus, thalamus, ventral midbrain, raphe, cerebellum and precerebellar system. This study represents the first detailed analysis of BCATc mRNA expression in the developing mouse brain.

Viability and neuronal differentiation of neural stem cells encapsulated in silk fibroin hydrogel functionalized with an IKVAV peptide.

Three-dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin-based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV-modified silk fibroin is a promising material for brain tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Genipin-crosslinked gelatin-silk fibroin hydrogels for modulating the behaviour of pluripotent cells.

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin-crosslinked gelatin-silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin-crosslinked gelatin-silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin-crosslinked gelatin-silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright © 2014 John Wiley & Sons, Ltd.