Targeting pathological cells with senolytic drugs reduces seizures in neurodevelopmental mTOR-related epilepsy.

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated ß-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.

Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

OBJECTIVE: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells. METHODS: We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex. RESULTS: We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. INTERPRETATION: BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, yet it remains refractory to systemic therapy. Elimination of senescent cells has emerged as a promising new treatment approach against cancer. Here, we investigated the contribution of senescent cells to GBM progression. Senescent cells are identified in patient and mouse GBMs. Partial removal of p16Ink4a-expressing malignant senescent cells, which make up less than 7 % of the tumor, modifies the tumor ecosystem and improves the survival of GBM-bearing female mice. By combining single cell and bulk RNA sequencing, immunohistochemistry and genetic knockdowns, we identify the NRF2 transcription factor as a determinant of the senescent phenotype. Remarkably, our mouse senescent transcriptional signature and underlying mechanisms of senescence are conserved in patient GBMs, in whom higher senescence scores correlate with shorter survival times. These findings suggest that senolytic drug therapy may be a beneficial adjuvant therapy for patients with GBM.

A recurrent point mutation in PRKCA is a hallmark of chordoid gliomas.

Chordoid glioma (ChG) is a characteristic, slow growing, and well-circumscribed diencephalic tumor, whose mutational landscape is unknown. Here we report the analysis of 16 ChG by whole-exome and RNA-sequencing. We found that 15 ChG harbor the same PRKCA D463H mutation. PRKCA encodes the Protein kinase C (PKC) isozyme alpha (PKCα) and is mutated in a wide range of human cancers. However the hot spot PRKCA D463H mutation was not described in other tumors. PRKCA D463H is strongly associated with the activation of protein translation initiation (EIF2) pathway. PKCαD463H mRNA levels are more abundant than wild-type PKCα transcripts, while PKCαD463H is less stable than the PCKαWT protein. Compared to PCKαWT, the PKCαD463H protein is depleted from the cell membrane. The PKCαD463H mutant enhances proliferation of astrocytes and tanycytes, the cells of origin of ChG. In conclusion, our study identifies the hallmark mutation for chordoid gliomas and provides mechanistic insights on ChG oncogenesis.

Injury-Induced Senescence Enables In Vivo Reprogramming in Skeletal Muscle.

In vivo reprogramming is a promising approach for tissue regeneration in response to injury. Several examples of in vivo reprogramming have been reported in a variety of lineages, but some including skeletal muscle have so far proven refractory. Here, we show that acute and chronic injury enables transcription-factor-mediated reprogramming in skeletal muscle. Lineage tracing indicates that this response frequently originates from Pax7+ muscle stem cells. Injury is associated with accumulation of senescent cells, and advanced aging or local irradiation further enhanced in vivo reprogramming, while selective elimination of senescent cells reduced reprogramming efficiency. The effect of senescence appears to be, at least in part, due to the release of interleukin 6 (IL-6), suggesting a potential link with the senescence-associated secretory phenotype. Collectively, our findings highlight a beneficial paracrine effect of injury-induced senescence on cellular plasticity, which will be important for devising strategies for reprogramming-based tissue repair.

Numb is required to prevent p53-dependent senescence following skeletal muscle injury.

Regeneration relies on coordinated action of multiple cell types to reconstitute the damaged tissue. Here we inactivate the endocytic adaptor protein Numb in skeletal muscle stem cells prior to chronic or severe muscle injury in mice. We observe two types of senescence in regenerating muscle; a transient senescence in non-myogenic cells of control and Numb mutant mice that partly depends on INK4a/ARF activity, and a persistent senescence in myogenic cells lacking Numb. The senescence levels of Numb-deficient muscle is reduced to wild type levels by an anti-oxidant treatment or p53 ablation, resulting in functional rescue of the regenerative potential in Numb mutants. Ex vivo experiments suggest that Numb-deficient senescent cells recruit macrophages to sustain inflammation and drive fibrosis, two hallmarks of the impaired muscle regeneration in Numb mutants. These findings provide insights into previously reported developmental and oncogenic senescence that are also differentially regulated by p53.

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4.

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4Cre and Mrf4nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.

Numb promotes an increase in skeletal muscle progenitor cells in the embryonic somite.

Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3(+)/Pax7(+) somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression.

Asymmetric cell divisions and asymmetric cell fates.

The regulation of self-renewal, cell diversity, and differentiation can occur by modulating symmetric and asymmetric cell divisions. Remarkably, asymmetric cell divisions can arise through multiple processes in which molecules in the cytoplasm and nucleus, as well as template "immortal" DNA strands, can segregate to one daughter cell during cell division. Explaining how these events direct distinct daughter cell fates is a major challenge to understanding how the organism is assembled and maintained for a lifetime. Numerous technical issues that are associated with assessing how distinct cell fates are executed in vivo have resulted in divergent interpretations of experimental findings. This review addresses some of these points and considers different developmental model systems that attempt to investigate how cell fate decisions are determined, as well as the molecules that guide these choices.

Early mouse caudal development relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways.

The progressive generation of embryonic trunk structures relies on the proper patterning of the caudal epiblast, which involves the integration of several signalling pathways. We have investigated the function of retinoic acid (RA) signalling during this process. We show that, in addition to posterior mesendoderm, primitive streak and node cells transiently express the RA-synthesizing enzyme Raldh2 prior to the headfold stage. RA-responsive cells (detected by the RA-activated RARE-lacZ transgene) are additionally found in the epiblast layer. Analysis of RA-deficient Raldh2(-/-) mutants reveals early caudal patterning defects, with an expansion of primitive streak and mesodermal markers at the expense of markers of the prospective neuroepithelium. As a result, many genes involved in neurogenesis and/or patterning of the embryonic spinal cord are affected in their expression. We demonstrate that RA signalling is required at late gastrulation stages for mesodermal and neural progenitors to respond to the Shh signal. Whole-embryo culture experiments indicate that the proper response of cells to Shh requires two RA-dependent mechanisms: (1) a balanced antagonism between Fgf and RA signals, and (2) a RA-mediated repression of Gli2 expression. Thus, an interplay between RA, Fgf and Shh signalling is likely to be an important mechanism underpinning the tight regulation of caudal embryonic development.

Combinatorial signalling controls Neurogenin2 expression at the onset of spinal neurogenesis.

A central issue during embryonic development is to define how different signals cooperate in generating unique cell types. To address this issue, we focused on the function and the regulation of the proneural gene Neurogenin2 (Neurog2) during early mouse spinal neurogenesis. We showed that Neurog2 is first expressed in cells within the neural plate anterior to the node from the 5 somite-stage. The analysis of Neurog2 mutants established a role for this gene in triggering neural differentiation during spinal cord elongation. We identified a 798 base pair enhancer element (Neurog2-798) upstream of the Neurog2 coding sequence that directs the early caudal expression of Neurog2. Embryo culture experiments showed that Retinoic Acid (RA), Sonic hedgehog (Shh) and Fibroblast Growth Factor signals act in concert on this enhancer to control the spatial and temporal induction of Neurog2. We further demonstrated by transgenesis that two RA response elements and a Gli binding site within the Neurog2-798 element are absolutely required for its activity, strongly suggesting that the regulation of Neurog2 early expression by RA and Shh signals is direct. Our data thus support a model where signal integration at the level of a single enhancer constitutes a key mechanism to control the onset of neurogenesis.

Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons.

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.

Stat3 signaling is present and active during development of the central nervous system and eye of vertebrates.

Stat3, a member of the signal transducer and activator of transcription (STAT) family, plays a central role in mediating cell growth, differentiation, and survival signals. In this report, we show that Stat3 immunoreactivity was localized to specific regions in the developing mouse brain, neural tube, and eye from embryonic day 10.5 to postnatal day 0. The active form of Stat3 protein, which is phosphorylated on tyrosine 705 (pYStat3), was also found in the developing neural tube with more restricted distribution. An in ovo chick embryo electroporation assay showed that the endogenous chick Stat3 could drive consensus sis-inducible element-directed reporter gene expression. These results demonstrate that the active Stat3 protein is present and might play a role during the development of the central nervous system and eye.

Direct and concentration-dependent regulation of the proneural gene Neurogenin2 by Pax6.

Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. We show that Pax6 function is both required and sufficient to activate this enhancer, and we identify one evolutionary conserved sequence in the E1 element with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.

Notch activity is required to maintain floorplate identity and to control neurogenesis in the chick hindbrain and spinal cord.

Notch signalling plays a major role in many invertebrate and vertebrate patterning systems. In this paper, we use high-titre, non-replicative pseudotype viruses to show that the two Notch ligands, Delta1 and Serrate1 (Jagged1), have differing activities in the developing chick spinal cord and hindbrain. In the walls of the neural tube, Serrate1 appears not to affect neurogenesis, in contrast to Delta1 which mediates lateral inhibition as elsewhere in the nervous system. In the floorplate we find that there is also a requirement for Notch, but with a different type of dependence on the two Notch ligands: cells with a floorplate character are lost when Notch activity is blocked with dominant-negative, truncated forms of either Delta1 or Serrate1. Our results are consistent with ligand-receptor specificity within the Notch signalling pathway, Serrate1 recognising selectively Notch2 (which is expressed in the floorplate), and Delta1 acting on both Notch2 and Notch1 (which is expressed in the walls of the neural tube).

The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system.

Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2), the enzyme responsible for early embryonic retinoic acid synthesis, is embryonic lethal because of defects in early heart morphogenesis. Transient maternal RA supplementation from E7.5 to (at least) E8.5 rescues most of these defects, but the supplemented Raldh2(-/-) mutants die prenatally, from a lack of septation of the heart outflow tract (Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Development 128, 1019-1031). We have investigated the developmental basis for this defect, and found that the RA-supplemented Raldh2(-/-) embryos exhibit impaired development of their posterior (3rd-6th) branchial arch region. While the development of the first and second arches and their derivatives, as well as the formation of the first branchial pouch, appear to proceed normally, more posterior pharyngeal pouches fail to form and the pharyngeal endoderm develops a rudimentary, pouch-like structure. All derivatives of the posterior branchial arches are affected. These include the aortic arches, pouch-derived organs (thymus, parathyroid gland) and post-otic neural crest cells, which fail to establish segmental migratory pathways and are misrouted caudally. Patterning and axonal outgrowth of the posterior (9th-12th) cranial nerves is also altered. Vagal crest deficiency in Raldh2(-/-) mutants leads to agenesis of the enteric ganglia, a condition reminiscent of human Hirschprung's disease. In addition, we provide evidence that: (i) wildtype Raldh2 expression is restricted to the posteriormost pharyngeal mesoderm; (ii) endogenous RA response occurs in both the pharyngeal endoderm and mesoderm, and extends more rostrally than Raldh2 expression up to the 2nd arch; (iii) RA target genes (Hoxa1, Hoxb1) are downregulated in both the pharyngeal endoderm and mesoderm of mutant embryos. Thus, RALDH2 plays a crucial role in producing RA required for pharyngeal development, and RA is one of the diffusible mesodermal signals that pattern the pharyngeal endoderm.

A floor plate enhancer of the zebrafish netrin1 gene requires Cyclops (Nodal) signalling and the winged helix transcription factor FoxA2.

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3beta, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2-given its importance for midline development also in the mouse-may be one such component.