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Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH.

Rapidity and specificity are characteristic features of proteolysis mediated by the ubiquitin-proteasome system. Therefore, the UPS is ideally suited for the remodeling of the embryonic stem cell proteome during the transition from pluripotent to differentiated states and its inverse, the generation of inducible pluripotent stem cells. The Trim-NHL family member LIN41 is among the first E3 ubiquitin ligases to be linked to stem cell pluripotency and reprogramming. Initially discovered in C. elegans as a downstream target of the let-7 miRNA, LIN41 is now recognized as a critical regulator of stem cell fates as well as the timing of neurogenesis. Despite being indispensable for embryonic development and neural tube closure in mice, the underlying mechanisms for LIN41 function in these processes are poorly understood. To better understand the specific contributions of the E3 ligase activity for the stem cell functions of LIN41, we characterized global changes in ubiquitin or ubiquitin-like modifications using Lin41-inducible mouse embryonic stem cells. The tumor suppressor protein p53 was among the five most strongly affected proteins in cells undergoing neural differentiation in response to LIN41 induction. We show that LIN41 interacts with p53, controls its abundance by ubiquitination and antagonizes p53-dependent pro-apoptotic and pro-differentiation responses. In vivo, the lack of LIN41 is associated with upregulation of Grhl3 and widespread caspase-3 activation, two downstream effectors of p53 with essential roles in neural tube closure. As Lin41-deficient mice display neural tube closure defects, we conclude that LIN41 is critical for the regulation of p53 functions in cell fate specification and survival during early brain development.

Beginning with their discovery in the context of stem cell fate choice in Caenorhabditis elegans, the microRNA (miRNA) let-7 and the RNA-binding protein Lin28 have been recognized as a regulatory pair with far-reaching impact on stem cell behavior in a wide range of organisms and tissues, including the mammalian brain. In this review, we describe molecular interactions between Lin28 and let-7 and the biological role that each plays in implementing stem cell programs that either maintain stem cell self-renewal and plasticity or drive lineage commitment and differentiation. For Lin28, considerable progress has been made in defining let-7-dependent and let-7-independent functions in the maintenance of pluripotency, somatic cell reprogramming, tissue regeneration, and neural stem cell plasticity. For the pro-differentiation activity of let-7, we focus on emerging roles in mammalian neurogenesis and neuronal function. Specific targets and pathways for let-7 have been identified in embryonic and adult neurogenesis, including corticogenesis, retinal specification, and adult neurogenic niches. Special emphasis is given to examples of feedback and feedforward regulation, in particular within the miRNA biogenesis pathway.

Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimer’s disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7(−/−) fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.

The let-7 miRNA and its target gene Lin-28 interact in a regulatory circuit controlling pluripotency. We investigated an additional let-7 target, mLin41 (mouse homologue of lin-41), as a potential contributor to this circuit. We demonstrate the presence of mLin41 protein in several stem cell niches, including the embryonic ectoderm, epidermis and male germ line. mLin41 colocalized to cytoplasmic foci with P-body markers and the miRNA pathway proteins Ago2, Mov10 and Tnrc6b. In co-precipitation assays, mLin41 interacted with Dicer and the Argonaute proteins Ago1, Ago2 and Ago4. Moreover, we show that mLin41 acts as an E3 ubiquitin ligase in an auto-ubiquitylation assay and that mLin41 mediates ubiquitylation of Ago2 in vitro and in vivo. Overexpression and depletion of mLin41 led to inverse changes in the level of Ago2 protein, implicating mLin41 in the regulation of Ago2 turnover. mLin41 interfered with silencing of target mRNAs for let-7 and miR-124, at least in part by antagonizing Ago2. Furthermore, mLin41 cooperated with the pluripotency factor Lin-28 in suppressing let-7 activity, revealing a dual control mechanism regulating let-7 in stem cells.

miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.

Lin41/Trim71 is a heterochronic gene encoding a member of the Trim-NHL protein family, and is the original, genetically defined target of the microRNA let-7 in C. elegans. Both the LIN41 protein and multiple regulatory microRNA binding sites in the 3’ UTR of the messenger RNA (mRNA) are highly conserved from nematodes to humans. Functional studies have described essential roles for mouse LIN41 in embryonic stem cells, cellular reprogramming and the timing of embryonic neurogenesis. We have used a new gene trap mouse line deficient in Lin41 to characterize Lin41 expression during embryonic development and in the postnatal central nervous system (CNS). In the embryo, Lin41 is required for embryonic viability and neural tube closure. Nevertheless, neurosphere assays suggest that Lin41 is not required for adult neurogenesis. Instead, we show that Lin41 promoter activity and protein expression in the postnatal CNS is restricted to ependymal cells lining the walls of the four ventricles. We use ependymal cell culture to confirm reestablishment of Lin41 expression during differentiation of ependymal progenitors to post-mitotic cells possessing motile cilia. Our results reveal that terminally differentiated ependymal cells express Lin41, a gene to date associated with self-renewing stem cells.

miR-128, a brain-enriched microRNA, has been implicated in the control of neurogenesis and synaptogenesis but its potential roles in intervening processes have not been addressed. We show that post-transcriptional mechanisms restrict miR-128 accumulation to post-mitotic neurons during mouse corticogenesis and in adult stem cell niches. Whereas premature miR-128 expression in progenitors for upper layer neurons leads to impaired neuronal migration and inappropriate branching, sponge-mediated inhibition results in overmigration. Within the upper layers, premature miR-128 expression reduces the complexity of dendritic arborization, associated with altered electrophysiological properties. We show that Phf6, a gene mutated in the cognitive disorder Börjeson-Forssman-Lehmann syndrome, is an important regulatory target for miR-128. Restoring PHF6 expression counteracts the deleterious effect of miR-128 on neuronal migration, outgrowth and intrinsic physiological properties. Our results place miR-128 upstream of PHF6 in a pathway vital for cortical lamination as well as for the development of neuronal morphology and intrinsic excitability. DOI: http://dx.doi.org/10.7554/eLife.04263.001, eLife digest The unique capabilities of the mammalian brain depend on the patterns formed by spatial arrangements and connections between millions (sometimes billions) of electrically active cells called neurons, and on the connections between these neurons. During the development of the cortex, the largest part of the brain, neurons are born in stem cell areas that lie deep inside the brain, and these newly made neurons then migrate outwards to their final positions close to the surface of the adult brain. Franzoni et al. have examined how two molecules, a small RNA called miR-128 and a protein called PHF6, control when and how neurons migrate through the cortex and then grow to form connections with other neurons as they mature. Mutations that disrupt PHF6 can cause intellectual disabilities, and one possible reason for this is that PHF6 is needed to ensure that the neurons migrate to the correction location. Franzoni et al. now show that miR-128 can reduce the production of PHF6 and is therefore responsible for controlling when and where PHF6 is active. Studying miR-128 in detail, they show that although an inactive precursor form of miR-128 is present in stem cells and migrating neurons, the active form of miR-128 is only found in neurons that have already reached their final position in the cortex. Franzoni et al. used genetic methods to override the switch that controls when miR-128 becomes active. When the amount of miR-128 was artificially reduced, the neurons migrated too far. Artificially increasing the amount of miR-128 had the opposite effect: both the movement of the neurons and, later, their growth were defective. PHF6 was the key to these effects: if PHF6 levels were kept close to normal, miR-128 could no longer interfere with the movement and growth of the neurons. Further work will be required to better understand how miR-128 is turned off and on, and how PHF6 acts to control neuronal movement and growth. DOI: http://dx.doi.org/10.7554/eLife.04263.002

The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.

The NF-kappaB precursor p105 has dual functions: cytoplasmic retention of attached NF-kappaB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signalling processes that are known to activate NF-kappaB p50-p65. We propose a model that p105 is inducibly degraded, and that its degradation liberates sequestered NF-kappaB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl-3. We show that TNFalpha, IL-1beta or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-alpha-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaBalpha, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-alpha stimulation, but only when the IKK phospho-acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super-repressor of IKK-induced NF-kappaB transcriptional activity. Thus, the known NF-kappaB stimuli not only cause nuclear accumulation of p50-p65 heterodimers but also of Bcl-3-p50 and perhaps further transcription activator complexes which are formed upon IKK-mediated p105 degradation.

Background & Aims Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. Methods Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. Results TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. Conclusions Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Innate immune receptors represent an evolutionarily ancient system that allows organisms to detect and rapidly respond to pathogen- and host-derived factors. TLRs are predominantly expressed in immune cells and mediate such a response. Although this class of pattern recognition receptors is involved in CNS disorders, the knowledge of ligands leading to activation of TLRs and to subsequent CNS damage is limited. We report in this study that ssRNA causes neurodegeneration and neuroinflammation dependent on TLR7 in the CNS. TLR7 is not only expressed in microglia, the major immune cells of the brain, but also in neurons of the CNS. Extracellularly delivered ssRNA40, an oligoribonucleotide derived from HIV and an established ligand of TLR7, induces neuronal cell death dependent on TLR7 and the central adapter molecule MyD88 in vitro. Activation of caspase-3 is involved in neuronal damage mediated by TLR7. This cell-autonomous neuronal cell death induced by ssRNA40 is amplified in the presence of microglia that mount an inflammatory response to ssRNA40 through TLR7. Intrathecal administration of ssRNA40 causes widespread neurodegeneration in wild-type but not in TLR7−/− mice, confirming that neuronal cell death induced by ssRNA40 through TLR7 occurs in vivo. Our results point to a possible mechanism through which extracellularly delivered ssRNA contributes to CNS damage and determine an obligatory role for TLR7 in this pathway.

Trim-NHL proteins are defined by RING, B-Box and Coiled-coil protein motifs (referred to collectively as the Trim domain) coupled to an NHL domain. The C. elegans, D. melanogaster, mouse and human Trim-NHL proteins are potential and in several cases confirmed, E3 ubiquitin ligases. Current research is focused on identifying targets and pathways for Trim-NHL-mediated ubiquitination and in assessing the contribution of the NHL protein-protein interaction domain for function and specificity. Several Trim-NHL proteins were discovered in screens for developmental genes in model organisms; mutations in one of the family members, Trim32, cause developmental disturbances in humans. In most instances, mutations that alter protein function map to the NHL domain. The NHL domain is a scaffold for the assembly of a translational repressor complex by the Brat proto-oncogene, a well-studied family member in Drosophila. The link to translational control is common to at least four Trim-NHLs that associate with miRNA pathway proteins. So far, two have been shown to repress (Mei-P26 and Lin41) and two to promote (NHL-2, Trim32) miRNA-mediated gene silencing. In this chapter we will describe structure-function relations for each of the proteins and then focus on the lessons being learned from these proteins about miRNA functions in development and in stem cell biology.

Trim-NHL proteins are defined by RING, B-Box and Coiled-coil protein motifs (referred to collectively as the Trim domain) coupled to an NHL domain. The C. elegans, D. melanogaster, mouse and human Trim-NHL proteins are potential and in several cases confirmed, E3 ubiquitin ligases. Current research is focused on identifying targets and pathways for Trim-NHL-mediated ubiquitination and in assessing the contribution of the NHL protein-protein interaction domain for function and specificity. Several Trim-NHL proteins were discovered in screens for developmental genes in model organisms; mutations in one of the family members, Trim32, cause developmental disturbances in humans. In most instances, mutations that alter protein function map to the NHL domain. The NHL domain is a scaffold for the assembly of a translational repressor complex by the Brat proto-oncogene, a well-studied family member in Drosophila. The link to translational control is common to at least four Trim-NHLs that associate with miRNA pathway proteins. So far, two have been shown to repress (Mei-P26 and Lin41) and two to promote (NHL-2, Trim32) miRNA-mediated gene silencing. In this chapter we will describe structure-function relations for each of the proteins and then focus on the lessons being learned from these proteins about miRNA functions in development and in stem cell biology.

The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons.

MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. We have studied the regulation of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. We observed significant differences in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes we found marked lineage specificity for each of the miRNA in our study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir-124, mir-128). In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. mir-26 and mir-29 were more strongly expressed in astrocytes than neurons, others were more evenly distributed (mir-9, mir-125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir-125 and mir-128). miRNA-mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA were strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development.