Search

Recent advances in genetic testing technologies have revolutionised the identification of genetic abnormalities in early onset developmental and epileptic encephalopathies (DEEs). In this Review, we provide an update on the expanding landscape of genetic factors contributing to DEEs, encompassing over 800 reported genes. We focus on the cellular and molecular mechanisms driving epileptogenesis, with an emphasis on emerging therapeutic strategies and effective treatment options. We explore noteworthy, novel genes linked to DEE phenotypes, such as gBRAT-1 and GNAO1, and gene families such as GRIN and HCN. Understanding the network-level effects of gene variants will pave the way for potential gene therapy applications. Given the diverse comorbidities associated with DEEs, a multidisciplinary team approach is essential. Despite ongoing efforts and improved genetic testing, DEEs lack a cure, and treatment complexities persist. This Review underscores the necessity for larger international prospective studies focusing on both seizure outcomes and developmental trajectories., Competing Interests: Declaration of interests NS has served on scientific advisory boards for GW Pharma, BioMarin, Arvelle, Marinus, and Takeda; has received speaker honoraria from Eisai, BioMarin, Livanova, Sanofi; and has served as an investigator for Zogenix, Marinus, BioMarin, Union Chimique Belge (UCB), and Roche. MT has served on advisory boards for BioMarin and Biocodex; has received speaker honoraria from BioMarin, Biocodex, and Orion; and has served as an investigator for Zogenix, Marinus, BioMarin, UCB, and Roche. SB has served on advisory boards for Biocodex and has received speaker and consultant honoraria from Angelini, Biocodex, and Jazz Pharmaceutics. AA has received speaker and consultant honoraria from Biocodex, EISAI, Jazz Pharmaceutics, Sanofi, and UCB and is co-director of the European Consortium for Epilepsy Trials. JHC has received grants from Stoke Therapeutics, Ultragenyx, UCB, the National Institute for Health and Care Research, Great Ormond Street Hospital Children's Charity, LifeARC, the Waterloo Foundation, and Action Medical Research. JHC has also received honoraria payments from Biocodex, Nutricia, Jazz Pharmaceuticals, Nutricia, Takeda, and UCB, all of which have been paid to University College London. JMW is associate editor of Epilepsia and chief editor of the Paediatric Neurology subsection of Frontiers in Neurology and has served on advisory boards for Sanofi and Novartis. FV has received speaker fees from Zogenix, Neuraxpharm, Angelini, and Eisai and has served on advisory boards for Zogenix, Neuraxpharm, Angelini, and Eisai. SA has received honoraria for lectures from Biocodex, BioMarin, Eisai, Jazz Pharmaceuticals, Neuraxpharm, Nutricia, Stoke, UCB, and Zogenix; has been paid as a consultant for lectures by Biocodex, Encoded, Grintherapeutics, Jazz Pharmaceuticals, Neuraxpharm, Nutricia, Orion, Proveca, Supernus, Stoke, Takeda, UCB, and Xenon; and has been an investigator for clinical trials for Eisai, Proveca, Takeda, and UCB. RN has served as principal investigator in clinical trials for Novartis, Nutricia, Eisai, UCB, GW Pharma, and Livanova; has received consulting and lecturer honoraria from BioGene, BioMarin, Praxis, GW Pharma, Zogenix, Novartis, Nutricia, Stoke, Ionis, Targeon, Neuraxpharma, Takeda, Nutricia, Biocodex, Advicennes, and Eisai; and has received unrestricted research grants from Eisai, UCB, Livanova, and GW Pharma and academic research grants from the European Joint Programme on Rare Diseases (Horizons 2020). PC has served on a scientific advisory board for Novartis, has received speaker honoraria from Jazz Pharmaceuticals and ItalFarmaco, and has served as investigator for clinical trials for Novartis. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Dravet Syndrome (DS) is most often caused by heterozygous loss-of-function mutations in the voltage-gated sodium channel gene SCN1A (Na v 1.1), resulting in severe epilepsy and neurodevelopmental impairment thought to be cause by reduced interneuron excitability. However, recent studies in mouse models suggest that interneuron dysfunction alone does not completely explain all the cellular and network impairments seen in DS. Here, we investigated the development of the intrinsic, synaptic, and network properties of CA1 pyramidal cells in a DS model prior to the appearance of overt seizures. We report that CA1 pyramidal cell development is altered by heterozygous reduction of Scn1a, and propose that this is explained by a period of reduced intrinsic excitability in early postnatal life, during which Scn1a is normally expressed in hippocampal pyramidal cells. We also use a novel ex vivo model of homeostatic plasticity to show an instability in homeostatic response during DS epileptogenesis. This study provides evidence for the early effects of Scn1a haploinsufficiency in pyramidal cells in contributing to the pathophysiology of DS., Competing Interests: Declaration of competing interest No conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, are caused by obligate intracellular pathogens that have clinically comparable presentations. Diagnostics used in laboratories today are serologic assays and blood smear analyses, which have known diagnostic limits. This study evaluated the performance of a sample-to-answer direct real-time PCR laboratory-developed test for the multiplex qualitative detection of Anaplasma , Babesia , and Ehrlichia DNA in whole-blood specimens. Compared to two standard-of-care (SOC) methods, the DiaSorin tick-borne laboratory-developed test for Anaplasma detection demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 100% (95% CI, 0.80 to 1.0) and 89% (95% CI, 0.74 to 0.97), respectively with a discordant rate of 9.3% against microscopy. After discordant resolution, the NPA increased to 100%. For Babesia , the test demonstrated a PPA of 100% (95% CI, 0.90 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Compared to a SOC PCR method Anaplasma samples showed a PPA of 100% (95% CI, 0.66 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Ehrlichia results showed a PPA of 100% (95% CI, 0.69 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). The total percent agreement was 98% (95% CI, 0.95 to 0.99) with a κ statistic of 0.95 (95% CI, 0.90 to 0.99) or almost perfect agreement compared to SOC methods. This laboratory-developed test for detecting Anaplasma , Babesia , and Ehrlichia DNA provides rapid and reliable detection of tick-borne infections without nucleic acid extraction., Importance: This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance., Competing Interests: Materials for this study were supplied by DiaSorin Molecular.

Dravet syndrome is a severe epileptic encephalopathy, characterized by drug-resistant epilepsy, severe cognitive and behavioural deficits, with increased risk of sudden unexpected death (SUDEP). It is caused by haploinsufficiency of SCN1A gene encoding for the α-subunit of the voltage-gated sodium channel Nav1.1. Therapeutic approaches aiming to upregulate the healthy copy of SCN1A gene to restore its normal expression levels are being developed. However, whether Scn1a gene function is required only during a specific developmental time-window or, alternatively, if its physiological expression is necessary in adulthood is untested up to now. We induced Scn1a gene haploinsufficiency at two ages spanning postnatal brain development (P30 and P60) and compared the phenotypes of those mice to Scn1a perinatally induced mice (P2), recapitulating all deficits of Dravet mice. Induction of heterozygous Nav1.1 mutation at P30 and P60 elicited susceptibility to the development of both spontaneous and hyperthermia-induced seizures and SUDEP rates comparable to P2-induced mice, with symptom onset accompanied by the characteristic GABAergic interneuron dysfunction. Finally, delayed Scn1a haploinsufficiency induction provoked hyperactivity, anxiety and social attitude impairment at levels comparable to age matched P2-induced mice, while it was associated with a better cognitive performance, with P60-induced mice behaving like the control group. Our data show that maintenance of physiological levels of Nav1.1 during brain development is not sufficient to prevent Dravet symptoms and that long-lasting restoration of Scn1a gene expression would be required to grant optimal clinical benefit in patients with Dravet syndrome., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans., (© 2023. The Author(s).)

Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a  Stop/+ ) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Na v 1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells., (© 2022. The Author(s).)

In vivo genome editing tools, such as those based on CRISPR, have been increasingly utilized in both basic and translational neuroscience research. There are currently nine in vivo non-CNS genome editing therapies in clinical trials, and the pre-clinical pipeline of major biotechnology companies demonstrate that this number will continue to grow. Several biotechnology companies commercializing in vivo genome editing and modification technologies are developing therapies for CNS disorders with accompanying large partnering deals. In this review, the authors discuss the current genome editing and modification therapy pipeline and those in development to treat CNS disorders. The authors also discuss the technical and commercial limitations to translation of these same therapies and potential avenues to overcome these hurdles., Competing Interests: PL was employed by Hummingbird Ventures. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lubroth, Colasante and Lignani.)

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Na v 1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na v 1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Crohn's disease (CD) is a chronic inflammatory condition that can affect different portions of the gastrointestinal tract. Lymphatic drainage was demonstrated to be dysfunctional in CD pathogenesis, ultimately causing the failure of the resolution of intestinal inflammation. To investigate the molecular mechanisms underlying these dysfunctions, we isolated human intestinal lymphatic endothelial cells (HILECs) from surgical specimens of patients undergoing resection for complicated CD (CD HILEC) and from a disease-free margin of surgical specimens of patients undergoing resection for cancer (healthy HILEC). Both cell types underwent transcriptomic profiling, and their barrier functionality was tested using a transwell-based co-culture system between HILEC and lamina propria mononuclear cells (LPMCs). Results showed CD HILEC displayed a peculiar transcriptomic signature that highlighted mTOR signaling as an orchestrator of leukocyte trafficking through the lymphatic barrier of CD patients. Moreover, we demonstrated that LPMC transmigration through the lymphatic endothelium of patients with CD depends on the capability of mTOR to trigger interleukin 20 receptor subunit α (IL20RA)-mediated intracellular signaling. Conclusively, our study suggests that leukocyte trafficking through the intestinal lymphatic microvasculature can be controlled by modulating IL20RA, thus leading to the resolution of chronic inflammation in patients with CD.

In recent years, the need to derive sources of specialized cell types to be employed for cell replacement therapies and modeling studies has triggered a fast acceleration of novel cell reprogramming methods. In particular, in neuroscience, a number of protocols for the efficient differentiation of somatic or pluripotent stem cells have been established to obtain a renewable source of different neuronal cell types. Alternatively, several neuronal populations have been generated through direct reprogramming/transdifferentiation, which concerns the conversion of fully differentiated somatic cells into induced neurons. This is achieved through the forced expression of selected transcription factors (TFs) in the donor cell population. The reprogramming cocktail is chosen after an accurate screening process involving lists of TFs enriched into desired cell lineages. In some instances, this type of studies has revealed the crucial role of TFs whose function in the differentiation of a given specific cell type had been neglected or underestimated. Herein, we will speculate on how the in vitro studies have served to better understand physiological mechanisms of neuronal development in vivo .

During cerebral cortex development, neural progenitors are required to elaborate a variety of cell differentiation signals to which they are continuously exposed. RA acid is a potent inducer of neuronal differentiation as it was found to influence cortical development. We report herein that TBR2, a transcription factor specific to Intermediate (Basal) Neural Progenitors (INPs), represses activation of the RA responsive element and expression of RA target genes in cell lines. This repressive action on RA signaling was functionally confirmed by the decrease of RA-mediated neuronal differentiation in neural stem cells stably overexpressing TBR2. In vivo mapping of RA activity in the developing cortex indicated that RA activity is detected in radial glial cells and subsequently downregulated in INPs, revealing a fine cell-type specific regulation of its signaling. Thus, TBR2 might be a molecular player in opposing RA signaling in INPs. Interestingly, this negative regulation is achieved at least in part by directly repressing the critical nuclear RA co-factor ZFP423. Indeed, we found ZFP423 to be expressed in the developing cortex and promote RA-dependent neuronal differentiation. These data indicate that TBR2 contributes to suppressing RA signaling in INPs, thereby enabling them to re-enter the cell cycle and delay neuronal differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Basal rates of autophagy can be markedly accelerated by environmental stresses. Recently, autophagy has been involved in cancer-induced muscle wasting. Aim of this study has been to evaluate if autophagy is induced in the skeletal muscle of cancer patients. The expression (mRNA and protein) of autophagic markers has been evaluated in intraoperative muscle biopsies. Beclin-1 protein levels were increased in cachectic cancer patients, suggesting autophagy induction. LC3B-I protein levels were not significantly modified. LC3B-II protein levels were significantly increased in cachectic cancer patients suggesting either increased autophagosome formation or reduced autophagosome turnover. Conversely, p62 protein levels were increased in cachectic and non-cachectic cancer patients, suggesting impaired autophagosome clearance. As for mitophagy, both Bnip3 and Nix/Bnip3L show a trend to increase in cachectic patients. In the same patients, Parkin levels significantly increased, while PINK1 was unchanged. At gene level, Beclin-1, p-62, BNIP3, NIX/BNIP3L and TFEB mRNAs were not significantly modulated, while LC3B and PINK1 mRNA levels were increased and decreased, respectively, in cachectic cancer patients. Autophagy is induced in the skeletal muscle of cachectic cancer patients, although autophagosome clearance appears to be impaired. Further studies should evaluate whether modulation of autophagy could represent a relevant therapeutic strategy in cancer cachexia.

Introduction: Non-arteritic anterior ischemic optic neuropathy (NAION) is one of the most widespread visually disabling diseases in the middle-aged and elderly population. It typically presents as acute painless unilateral vision loss in patients over 50 years of age. The fellow eye of NAION patients is often sequentially affected. Involvement of the second eye occurs within three years in approximately 45%-50% of patients. Currently there is no generally accepted treatment for NAION but a number of medical and surgical therapies have been proposed., Report of a Case: This is a case of non-contemporary bilateral non-arteritic anterior ischemic optic neuropathy (NAION) in a 66-year old woman treated with hyperbaric oxygen (HBO2) therapy after ineffective systemic corticosteroid therapy. Visual acuity (VA), visual evoked potentials (VEP) findings, perimetric examination results and angiographic images were recorded and analyzed before and after hyperbaric oxygen treatment., Discussion: After several months from the optic nerve vascular injury, VA, VEP values, perimetric examination results and angiographic images revealed a very important recovery. These results maintained stable during the follow-up at about nine months. HBO2 therapy has been revealed to be a safe and efficacious adjunctive therapy, even after many months after the injury. While this case is promising, double-blind randomized controlled trials will be necessary to prove the efficacy of HBO2 in the treatment of NAION.

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During development, neural progenitor cells (NPCs) that are capable of self-renewing maintain a proliferative cellular pool while generating all differentiated neural cell components. Although the genetic network of transcription factors (TFs) required for neural specification has been well characterized, the unique set of histone modifications that accompanies this process has only recently started to be investigated. In vitro neural differentiation of pluripotent stem cells is emerging as a powerful system to examine epigenetic programs. Deciphering the histone code and how it shapes the chromatin environment will reveal the intimate link between epigenetic changes and mechanisms for neural fate determination in the developing nervous system. Furthermore, it will offer a molecular framework for a stringent comparison between native and induced neural stem cells (iNSCs) generated by direct neural cell conversion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinson's disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches.

The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus QuickFISH, for the direct detection of Staphylococcus species from positive blood culture bottles was evaluated in a multicenter clinical study. The method utilizes a microscope slide with predeposited positive- and negative-control organisms and a self-reporting 15-min hybridization step, which eliminates the need for a wash step. Five clinical laboratories tested 722 positive blood culture bottles containing gram-positive cocci in clusters. The sensitivities for detection of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were 99.5% (217/218) and 98.8% (487/493), respectively, and the combined specificity of the assay was 89.5% (17/19). The combined positive and negative predictive values of the assay were 99.7% (696/698) and 70.8% (17/24), respectively. Studies were also performed on spiked cultures to establish the specificity and performance sensitivity of the method. Staphylococcus QuickFISH has a turnaround time (TAT) of <30 min and a hands-on time (HOT) of <5 min. The ease and speed of the method have the potential to improve the accuracy of therapeutic intervention by providing S. aureus/CoNS identification simultaneously with Gram stain results.

Little is known about how, during its formidable expansion in development and evolution, the cerebral cortex is able to maintain the correct balance between excitatory and inhibitory neurons. In fact, while the former are born within the cortical primordium, the latter originate outward in the ventral pallium. Therefore, it remains to be addressed how these two neuronal populations might coordinate their relative amounts in order to build a functional cortical network. Here, we show that Tbr2-positive cortical intermediate (basal) neuronal progenitors (INPs) dictate the migratory route and control the amount of subpallial GABAergic interneurons in the subventricular zone (SVZ) through a non-cell-autonomous mechanism. In fact, Tbr2 interneuron attractive activity is moderated by Cxcl12 chemokine signaling, whose forced expression in the Tbr2 mutants can rescue, to some extent, SVZ cell migration. We thus propose that INPs are able to control simultaneously the increase of glutamatergic and GABAergic neuronal pools, thereby creating a simple way to intrinsically balance their relative accumulation.

The homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development.

The Arx transcription factor is expressed in the developing ventral telencephalon and subsets of its derivatives. Mutation of human ARX ortholog causes neurological disorders including epilepsy, lissencephaly, and mental retardation. We have isolated the mouse Arx endogenous enhancer modules that control its tightly compartmentalized forebrain expression. Interestingly, they are scattered downstream of its coding region and partially included within the introns of the downstream PolA1 gene. These enhancers are ultraconserved noncoding sequences that are highly conserved throughout the vertebrate phylum. Functional characterization of the Arx GABAergic enhancer element revealed its strict dependence on the activity of Dlx transcription factors. Dlx overexpression induces ectopic expression of endogenous Arx and its isolated enhancer, whereas loss of Dlx expression results in reduced Arx expression, suggesting that Arx is a key mediator of Dlx function. To further elucidate the mechanisms involved, a combination of gain-of-function studies in mutant Arx or Dlx tissues was pursued. This analysis provided evidence that, although Arx is necessary for the Dlx-dependent promotion of interneuron migration, it is not required for the GABAergic cell fate commitment mediated by Dlx factors. Although Arx has additional functions independent of the Dlx pathway, we have established a direct genetic relationship that controls critical steps in the development of telencephalic GABAergic neurons. These findings contribute elucidating the genetic hierarchy that likely underlies the etiology of a variety of human neurodevelopmental disorders.

The GAS multigene family of Saccharomyces cerevisiae is composed of five paralogs (GAS1 to GAS5). GAS1 is the only one of these genes that has been characterized to date. It encodes a glycosylphosphatidylinositol-anchored protein functioning as a beta(1,3)-glucan elongase and required for proper cell wall assembly during vegetative growth. In this study, we characterize the roles of the GAS2 and GAS4 genes. These genes are expressed exclusively during sporulation. Their mRNA levels showed a peak at 7 h from induction of sporulation and then decreased. Gas2 and Gas4 proteins were detected and reached maximum levels between 8 and 10 h from induction of sporulation, a time roughly coincident with spore wall assembly. The double null gas2 gas4 diploid mutant showed a severe reduction in the efficiency of sporulation, an increased permeability of the spores to exogenous substances, and production of inviable spores, whereas the single gas2 and gas4 null diploids were similar to the parental strain. An analysis of spore ultrastructure indicated that the loss of Gas2 and Gas4 proteins affected the proper attachment of the glucan to the chitosan layer, probably as a consequence of the lack of coherence of the glucan layer. The ectopic expression of GAS2 and GAS4 genes in a gas1 null mutant revealed that these proteins are redundant versions of Gas1p specialized to function in a compartment at a pH value close to neutral.