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PDF file - 65K, Supplementary Figure 6. A schematic drawing of full length N1ICD in mouse, N1ICD allele analyzed in this study, and the ACTB-Notch1 allele previously published (Yang et al., 2004) and analyzed in Supplementary Figure 6.

PDF file - 133K, Supplementary Figure 3. Reduced p53 dose rescues precocious GFAP expression in the N1ICD;hGFAP-cre brains.

PDF file - 84K, Supplementary Figure 2. Reduced p53 dose rescues brain sizes in N1ICD;hGFAP-cre transgenic mice.

While activation of the Notch pathway is observed in many human cancers, it is unknown whether elevated Notch1 expression is sufficient to initiate tumorigenesis in most tissues. To test the oncogenic potential of Notch1 in solid tumors, we expressed an activated form of Notch1 (N1ICD) in the developing mouse brain. N1ICD;hGFAP-cre mice were viable but developed severe ataxia and seizures, and died by weaning age. Analysis of transgenic embryo brains revealed that N1ICD expression induced p53-dependent apoptosis. When apoptosis was blocked by genetic deletion of p53, 30% to 40% of N1ICD;GFAP-cre;p53+/− and N1ICD;GFAP-cre;p53−/− mice developed spontaneous medulloblastomas. Interestingly, N1ICD-induced medulloblastomas most closely resembled the sonic hedgehog subgroup of human medulloblastoma at the molecular level. Surprisingly, N1ICD-induced tumors do not maintain high levels of the Notch pathway gene expression, except for Notch2, showing that initiating oncogenic events may not be decipherable by analyzing growing tumors in some cases. In summary, this study shows that Notch1 has an oncogenic potential in the brain when combined with other oncogenic hits, such as p53 loss, and provides a novel mouse model of medulloblastoma. Cancer Res; 73(17); 5381–90. ©2013 AACR.

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PDF file - 158K, Supplementary Figure 1. Reduced p53 dose rescues apoptosis in N1ICD;hGFAP-cre brains.

PDF file - 199K, Supplementary Figure 5. Increased apoptosis and reduced proliferation of neural precursor cells in ACTB-N1ICD brains.

PDF file - 84K, Supplementary Figure 4. N1ICD induced medulloblastomas.

The authors show that Netrin-1-Unc5B signaling controls blood-brain barrier integrity by maintaining Wnt/b-catenin signaling and that delivery of antibodies blocking Netrin-1 binding to Unc5B causes transient and size-selective BBB breakdown. Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/beta-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/beta-catenin signaling, and beta-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/beta-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.

Significance Understanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNA Arg UCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.

Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we showed that the endothelial Netrin1 receptor Unc5B controls BBB integrity by maintaining Wnt/β–catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice led to region and size-selective BBB opening. Loss of Unc5B decreased BBB Wnt/β–catenin signaling, and β–catenin overexpression rescued Unc5B mutant BBB defects. Mechanistically, Netrin1 enhanced Unc5B interaction with the Wnt co-receptor LRP6, induced its phosphorylation and activated Wnt/β–catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin1 binding to Unc5B caused a transient disruption of Wnt signaling and BBB breakdown, followed by neurovascular barrier resealing. These data identify Netrin-Unc5B signaling as a novel regulator of BBB integrity with potential therapeutic utility for CNS diseases.

ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) β-oxidation pathway in peroxisomes and leads to H(2)O(2) production. Unexpectedly, dACOX1 is mostly expressed and required in glia, and loss of dACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an anti-oxidant suppressed the p.N237S induced neurodegeneration. In summary, both loss and gain of ACOX1 leads to glial and neuronal loss, but different mechanisms are at play and require different treatments.

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD) and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and thein vivodefects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factorsPeloandHbs1lare critical for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, the translatome and cerebellar development were observed upon deletion of the NMD factorUpf2.These data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses.

Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of a tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here, we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant GTPases function in the same pathway to mitigate ribosome pausing. As observed in Gtpbp2-/- mice (Ishimura et al., 2016), GCN2-mediated activation of the integrated stress response (ISR) was apparent in the Gtpbp1-/- brain. We observed decreased mTORC1 signaling which increased neuronal death, whereas ISR activation was neuroprotective. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective elongation.

Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of an individual tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant translational GTPases function in the same pathway to mitigate ribosome pausing. Ribosome stalling in the mutant brain led to activation of the integrated stress response (ISR) mediated by GCN2 and decreased mTORC1 signaling. However, in contrast to the ISR, which enhanced neuron survival, reduced mTORC1 signaling increased neuronal death. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective translation elongation.

Netrins are secreted proteins that direct cell migration and axon extension in the developing CNS and are essential for normal neural development. In the mature CNS, netrin-1 is expressed by neurons and oligodendrocytes and implicated in the stability of axo-oligodendroglial paranodal junctions. Here we report that the netrin receptor UNC5B is highly expressed by mature oligodendrocytes and enriched at paranodes. We demonstrate that paranodes become disorganized following conditional deletion of UNC5B in oligodendrocytes, with disruption of the interface between glial loops and detachment of glial loops from the axon. Examining axoglial domain segregation, Caspr1 and Kv1.1 disperse along the axon, internodes shorten, and the periodicity of compact myelin is reduced, indicating significant breakdown of myelin organization in UNC5B cKOs. Paranodal disruption and axoglial domain disorganization progressively worsen with age and a delay in motor learning develops specifically in aged animals that lack oligodendroglial UNC5B. We detect reduced amounts of oligodendroglial Claudin-11 and JAM-C proteins in UNC5B knockouts, suggesting that disruption of the specialized autotypic junctions between glial loops may underlie paranodal disorganization. Our findings reveal an essential contribution of oligodendroglial UNC5B at axoglial junctions that is required for the stability of mature myelin.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNAAla and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNAAla and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aarssti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

Errors during mRNA translation can lead to a reduction in the levels of functional proteins and an increase in deleterious molecules. Advances in next-generation sequencing have led to the discovery of rare genetic disorders, many caused by mutations in genes encoding the mRNA translation machinery, as well as to a better understanding of translational dynamics through ribosome profiling. We discuss here multiple neurological disorders that are linked to errors in tRNA aminoacylation and ribosome decoding. We draw on studies from genetic models, including yeast and mice, to enhance our understanding of the translational defects observed in these diseases. Finally, we emphasize the importance of tRNA, their associated enzymes, and the inextricable link between accuracy and efficiency in the maintenance of translational fidelity.

Dynamic regulation of mRNA translation initiation and elongation is essential for the survival and function of neural cells. Global reductions in translation initiation resulting from mutations in the translational machinery or inappropriate activation of the integrated stress response may contribute to pathogenesis in a subset of neurodegenerative disorders. Aberrant proteins generated by non-canonical translation initiation may be a factor in the neuron death observed in the nucleotide repeat expansion diseases. Dysfunction of central components of the elongation machinery, such as the tRNAs and their associated enzymes, can cause translational infidelity and ribosome stalling, resulting in neurodegeneration. Taken together, dysregulation of mRNA translation is emerging as a unifying mechanism underlying the pathogenesis of many neurodegenerative disorders.

Active Vitamin D sterols such as calcitriol and alfacalcidol are quite effective in the treatment of mineral bone disease secondary to chronic kidney disease. However, some children on peritoneal dialysis (PD) are resistant to oral formulations of active Vitamin D, and use of an intravenous formulation in such patients is inconvenient. In these children, intraperitoneal (IP) calcitriol has been shown to be effective in the treatment of secondary hyperparathyroidism. However, its use has declined. We report 2 children, aged 1 and 9.5 years, on chronic cycler PD with severe secondary hyperparathyroidism refractory to oral active Vitamin D who were successfully treated with IP calcitriol for a period of 12 and 4 months, respectively. We also discuss the published literature on the efficacy of IP calcitriol for treatment of secondary hyperparathyroidism and specific considerations for its use in PD patients.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aarssti mutant mice resulted in an increased production of serine-mischarged tRNAAla and degeneration of cerebellar Purkinje cells. By positional cloning, we identified Ankrd16, which acts epistatically with the Aarssti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific, ankyrin repeat-containing protein, binds directly to the catalytic domain of AlaRS. Serine misactivated by AlaRS is captured by lysine side chains of ANKRD16, preventing the charging of serine adenylates to tRNAAla and precluding serine misincorporation in nascent peptides. Deletion of Ankrd16 in the Aarssti/sti brain causes widespread protein aggregation and neuron loss. These results identify a novel amino acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery essential for preventing severe pathologies that arise from defects in editing.

Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in theClcc1gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown ofClcc1expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated inClcc1-deficient granule cellsin vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death.

Misfolded proteins are an emerging hallmark of cardiac diseases. Although some misfolded proteins, such as desmin, are associated with mutations in the genes encoding these disease-associated proteins, little is known regarding more general mechanisms that contribute to the generation of misfolded proteins in the heart. Reduced translational fidelity, caused by a hypomorphic mutation in the editing domain of alanyl-tRNA synthetase (AlaRS), resulted in accumulation of misfolded proteins in specific mouse neurons. By further genetic modulation of the editing activity of AlaRS, we generated mouse models with broader phenotypes, the severity of which was directly related to the degree of compromised editing. Severe disruption of the editing activity of AlaRS caused embryonic lethality, whereas an intermediate reduction in AlaRS editing efficacy resulted in ubiquitinated protein aggregates and mitochondrial defects in cardiomyocytes that were accompanied by progressive cardiac fibrosis and dysfunction. In addition, autophagic vacuoles accumulated in mutant cardiomyocytes, suggesting that autophagy is insufficient to eliminate misfolded proteins. These findings demonstrate that the pathological consequences of diminished tRNA synthetase editing activity, and thus translational infidelity, are dependent on the cell type and the extent of editing disruption, and provide a previously unidentified mechanism underlying cardiac proteinopathy.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars

Problems making proteins kills nerve cells Neurodegeneration is associated with a variety of different diseases, but its cellular roots are often obscure. Ishimura et al. find that mutant mice whose brain cells start to die rapidly soon after birth have lost the function of two vital cellular components (see the Perspective by Darnell). The first is a protein that releases stalled ribosomes stuck on messenger RNA (mRNA); the second is a transfer RNA (tRNA), which reads the code for arginine in the mRNA. This tRNA is expressed predominantly in the central nervous system. The lack of the tRNA leads to increased ribosomal stalling at arginine codons, which, when left uncorrected, blocks protein synthesis and proves fatal. Science , this issue p. 455 ; see also p. 378

Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2nmf205-/- mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNAArgUCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2nmf205-/- mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress. DOI: http://dx.doi.org/10.7554/eLife.14295.001, eLife digest Information stored in DNA is used to make proteins in a two-step process. First, the DNA is copied to make molecules of messenger ribonucleic acid (or messenger RNA for short). Next, machines called ribosomes use the messenger RNAs as templates to assemble chains of amino acids – the building blocks of proteins – in a process called translation. Another type of RNA molecule called transfer RNA carries each amino acid to the ribosomes. If a specific transfer RNA is not available for translation at the right time, the ribosome might stall as it moves along the messenger RNA. At this point, the ribosome needs to be restarted or it will fall off the mRNA without finishing the protein. In 2014, a group of researchers reported that certain types of brain cells are very sensitive to ribosome stalling, and tend to die if translation does not continue. A protein called GTPBP2 was shown to play an important role in restarting stalled ribosomes in these cells. Here, Ishimura, Nagy et al. – including some of the researchers from the earlier work – investigated the molecular pathways that ribosome stalling triggers in brain cells using mutant mice that lacked the GTPBP2 protein. The experiments show that ribosome stalling activates an enzyme known as GCN2, which was already known to sense other types of malfunctions in cellular processes. Ishimura, Nagy et al. also show that GCN2 triggers stress responses in the cells by activating a communication system called the ATF4 pathway. This pathway protects the cells from damage, and its absence results in more rapid cell deterioration and death. The next challenges are to understand the exact mechanism by which GCN2 senses stalled ribosomes, and to find out how ribosome stalling causes the death of brain cells. DOI: http://dx.doi.org/10.7554/eLife.14295.002

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin–ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways. DOI: http://dx.doi.org/10.7554/eLife.10841.001, eLife digest The ability of animals to walk and perform skilled movements depends on particular groups of muscles contracting in a coordinated manner. Muscles are activated by nerve cells called motor neurons found in the spinal cord. The connections between the motor neurons and muscles are established in the developing embryo. Each motor neuron produces a long projection called an axon whose growth is guided towards the target muscle by signal proteins. The motor neurons are exposed to many such signal proteins at the same time and it is not clear how they integrate all this information so that their axons target the correct muscles. Poliak, Morales et al. used a variety of genetic and biochemical approaches to study the formation of motor neuron and muscle connections in the limbs of mice and chicks. The experiments show that a signal protein called Netrin-1 is produced in the limbs of developing embryos and attracts the axons of some types of motor neurons and repels others. This is due to the motor neurons producing different types of receptor proteins to detect Netrin-1. Further experiments show that individual axons can combine information from attractive or repulsive Netrin-1 signals together with repulsive signals from another family of proteins called ephrins in a 'synergistic' manner. That is, the combined effect of both cues is stronger than their individual effects added together. This synergy involves ligand-dependent interactions between the Netrin-1 and ephrin receptor proteins, and the activation of a common enzyme. Poliak, Morales et al.’s findings reveal a new role for Netrin-1 in guiding the development of motor neurons in the limb. Future work will focus on further understanding the mechanism of synergy between Netrin-1 and ephrins. Netrin-1 and ephrins are also involved in the formation of blood vessels and many other developmental processes, so understanding how they work together would have a wide-reaching impact on research into human health and disease. DOI: http://dx.doi.org/10.7554/eLife.10841.002

The cerebellum receives its input from multiple precerebellar nuclei located in the brainstem and sends processed information to other brain structures via the deep cerebellar neurons. Guidance molecules that regulate the complex migrations of precerebellar neurons and the initial guidance of their leading processes have been identified. However, the molecules necessary for dorsal guidance of precerebellar axons to the developing cerebellum or for guidance of decussating axons of the deep nuclei are not known. To determine whetherUnc5cplays a role in the dorsal guidance of precerebellar and deep cerebellar axons, we studied axonal trajectories of these neurons inUnc5c−/−mice. Our results show thatUnc5cis expressed broadly in the precerebellar and deep cerebellar neurons.Unc5cdeletion disrupted long-range dorsal guidance of inferior olivary and pontine axons after crossing the midline. In addition, dorsal guidance of the axons from the medial deep cerebellar and external cuneate neurons was affected inUnc5c−/−mice, as were anterior migrations of pontine neurons. Coincident with the guidance defects of their axons, degeneration of neurons in the external cuneate nucleus and subdivisions of the inferior olivary nucleus was observed inUnc5c−/−mice. Lastly, transgenic expression ofUnc5cin deep neurons and pontine neurons by theAtoh1promoter rescued defects of the medial deep cerebellar and pontine axons observed inUnc5c−/−embryos, demonstrating thatUnc5cacts cell autonomously in the guidance of these axons. Our results suggest thatUnc5cplays a broad role in dorsal guidance of axons in the developing hindbrain.

During their migration, cerebellar granule cells switch from a tangential to a radial mode of migration. We have previously demonstrated that this involves the transmembrane semaphorin Sema6A. We show here that plexin-A2 is the receptor that controls Sema6A function in migrating granule cells. In plexin-A2-deficient (Plxna2(-/-)) mice, which were generated by homologous recombination, many granule cells remained in the molecular layer, as we saw in Sema6a mutants. A similar phenotype was observed in mutant mice that were generated by mutagenesis with N-ethyl-N-nitrosourea and had a single amino-acid substitution in the semaphorin domain of plexin-A2. We found that this mutation abolished the ability of Sema6A to bind to plexin-A2. Mouse chimera studies further suggested that plexin-A2 acts in a cell-autonomous manner. We also provide genetic evidence for a ligand-receptor relationship between Sema6A and plexin-A2 in this system. Using time-lapse video microscopy, we found that centrosome-nucleus coupling and coordinated motility were strongly perturbed in Sema6a(-/-) and Plxna2(-/-) granule cells. This suggests that semaphorin-plexin signaling modulates cell migration by controlling centrosome positioning.

In both invertebrates and vertebrates, UNC5 receptors facilitate chemorepulsion away from a Netrin source. Unlike most motor neurons in the embryonic vertebrate spinal cord, spinal accessory motor neuron (SACMN) cell bodies and their axons translocate along a dorsally directed trajectory away from the floor plate/ventral midline and toward the lateral exit point (LEP). We have recently shown that Netrin-1 and DCC are required for the migration of SACMN cell bodies, in vivo. These observations raised the possibility that vertebrate UNC5 proteins mediate the presumed repulsion of SACMN away from the Netrin-rich ventral midline. Here, we show that SACMN are likely to express UNC5A and UNC5C. Whereas SACMN development proceeds normally in UNC5A null mice, many SACMN cell bodies fail to migrate away from the ventral midline and inappropriately cluster in the ventrolateral spinal cord of mouse embryos lacking UNC5C. These results support an important role for UNC5C in SACMN development.

Icodextrin is a solution of glucose polymers developed to provide sustained ultrafiltration over an extended dwell. Our aim was to determine whether or not fill volume with icodextrin contributes to the ability to achieve ultrafiltration in children.The charts of all children on chronic peritoneal dialysis between January 2000 and July 2014 were screened for the use of an icodextrin day dwell. Data were extracted from the electronic chart and the HomeChoice™ Pro card and corrected for body surface area (BSA).Fifty children had an icodextrin day dwell. A linear correlation was found between the daytime fill volume and net ultrafiltration (p 0.001). More ultrafiltration was achieved with a fill volume above 550 ml/m(2) BSA (107 ± 75 ml/m(2) BSA) than with smaller fill volumes (-8 ± 99 ml; p = 0.004). Ultrafiltration was achieved in 88 % of children with a fill volume above 550 ml/m(2) BSA versus only 44 % of patients with a smaller fill volume (p = 0.001). Icodextrin was well tolerated.Our observations reveal that the larger the fill volume the higher the likelihood of achieving ultrafiltration with icodextrin and suggest that a minimum day dwell volume of 550 ml/m(2) BSA seems to facilitate ultrafiltration in children. To our knowledge this is the largest study addressing ultrafiltration with icodextrin in children.

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.

Unfolded proteins and other conditions affecting endoplasmic reticulum (ER) homeostasis cause ER stress. The cell reacts to ER stress by activation of the unfolded protein response (UPR), which induces profound changes in cellular metabolism including general translation attenuation, transcriptional upregulation of molecular chaperone genes, and activation of ER-associated degradation. However, prolonged or acute ER stress results in cell death. Recent progress suggests that ER stress and UPR play key roles in the immune response, diabetes, tumor growth under hypoxic conditions, and in some neurodegenerative diseases. Further research on ER stress and UPR will greatly enhance the understanding of these physiological and pathological processes, and provide novel avenues to potential therapies.

Magnetic resonance imaging and computer image analysis in human clinical studies effectively identify abnormal neuroanatomy in disease populations. As more mouse models of neurological disorders are discovered, such an approach may prove useful for translational studies. Here, we demonstrate the effectiveness of a similar strategy for mouse neuroscience studies by phenotyping mice with the cerebellar deficient folia (cdf) mutation. Usingin vivomultiple-mouse magnetic resonance imaging for increased throughput, we imaged groups ofcdfmutant, heterozygous, and wild-type mice and made an atlas-based segmentation of the structures in 15 individual brains. We then performed computer automated volume measurements on the structures. We found a reduced cerebellar volume in thecdfmutants, which was expected, but we also found a new phenotype in the inferior colliculus and the olfactory bulbs. Subsequent local histology revealed additional cytoarchitectural abnormalities in the olfactory bulbs. This demonstrates the utility of anatomical magnetic resonance imaging and semiautomated image analysis for detecting abnormal neuroarchitecture in mutant mice.

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a `waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore,netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5crcm mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.

Endoplasmic reticulum (ER) chaperones and ER stress have been implicated in the pathogenesis of neurodegenerative disorders, such as Alzheimer and Parkinson diseases, but their contribution to neuron death remains uncertain1,2. In this study, we establish a direct in vivo link between ER dysfunction and neurodegeneration. Mice homozygous with respect to the woozy (wz) mutation develop adult-onset ataxia with cerebellar Purkinje cell loss. Affected cells have intracellular protein accumulations reminiscent of protein inclusions in both the ER and the nucleus. In addition, upregulation of the unfolded protein response, suggestive of ER stress, occurs in mutant Purkinje cells. We report that the wz mutation disrupts the gene Sil1 that encodes an adenine nucleotide exchange factor of BiP3, a crucial ER chaperone4. These findings provide evidence that perturbation of ER chaperone function in terminally differentiated neurons leads to protein accumulation, ER stress and subsequent neurodegeneration.

Rett syndrome is caused by mutations in the gene encoding the transcriptional regulator MECP2. A new study demonstrates that cholesterol homeostasis is disrupted in Mecp2 mutant mice and suggests new therapeutic options for this disease.

Harlequin (Hq) mutant mice have progressive degeneration of terminally differentiated cerebellar and retinal neurons. We have identified the Hq mutation as a proviral insertion in the apoptosis-inducing factor (Aif) gene, causing about an 80% reduction in AIF expression. Mutant cerebellar granule cells are susceptible to exogenous and endogenous peroxide-mediated apoptosis, but can be rescued by AIF expression. Overexpression of AIF in wild-type granule cells further decreases peroxide-mediated cell death, suggesting that AIF serves as a free radical scavenger. In agreement, dying neurons in aged Hq mutant mice show oxidative stress. In addition, neurons damaged by oxidative stress in both the cerebellum and retina of Hq mutant mice re-enter the cell cycle before undergoing apoptosis. Our results provide a genetic model of oxidative stress-mediated neurodegeneration and demonstrate a direct connection between cell cycle re-entry and oxidative stress in the ageing central nervous system.

La Jolla, CA. Protein synthesis is vital for cell function and disturbances in translation are associated with cancer, neurological disorders and other diseases. Although regulation of translation is thought to largely occur at initiation, elongation, the reiterative addition of amino acids by the ribosome, is emerging as a translational phase that is also highly regulated. Transfer RNAs, the adaptor molecules essential for elongation are encoded by several hundreds of nuclear genes in higher eukaryotes suggesting great functional redundancy. Our work has defined epistatic interactions between mutations in a mammalian tRNA gene and a novel ribosome rescue factor, defining the importance of individual tRNA genes in translation and the pathologies that can arise from disruptions in this process. Citation Format: Susan L. Ackerman. Ribosome stalling and disease. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA13.

In higher eukaryotes, transfer RNAs (tRNAs) with the same anticodon are encoded by multiple nuclear genes, and little is known about how mutations in these genes affect translation and cellular homeostasis. Similarly, the surveillance systems that respond to such defects in higher eukaryotes are not clear. Here, we discover that loss of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, in mice with a mutation in a tRNA gene that is specifically expressed in the central nervous system causes ribosome stalling and widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor but also unmask the disease potential of mutations in nuclear-encoded tRNA genes.

Purkinje cells of different molecular phenotypes subdivide the cortex of the cerebellum both rostrocaudally into parasagittal bands and mediolaterally into transverse zones. Superimposed on the Purkinje cell compartmentation, the cerebellar cortex is pleated into a reproducible array of lobes and lobules. During cerebellar development, Purkinje cell bands are formed through the rostrocaudal dispersal of embryonic clusters, triggered primarily by a Reelin-dependent signaling pathway. In the naturally occurring mouse mutant cerebellar deficient folia (cdf), there is a failure of Purkinje cell dispersion that results in widespread Purkinje cell ectopia in the adult. The ectopia is restricted primarily to that subset of Purkinje cells that does not express zebrin II/aldolase C and that forms ectopic clusters in among the cerebellar nuclei. Most Purkinje cells that express zebrin II are located normally in a monolayer. Thus, the cerebellum of cdf mutants has a failure of Purkinje cell dispersion that is confined primarily to a zebrin II-negative (zebrin II−) subpopulation. Despite the Purkinje cell ectopia, the parasagittal band organization of the cerebellum is still clear. The shortening of the cortex is distributed evenly over all lobules, with the result that transverse expression boundaries are relocated with respect to the lobules and fissures. The number of Purkinje cells in the cdf/cdf cerebellum is similar to the number in littermate controls. Therefore, it appears that the lesion in cdf results in the failure of a zebrin II− Purkinje cell subset to disperse either due to a cell intrinsic defect or due to an abnormal interaction between the Purkinje cells and either granule cells or afferent inputs. J. Comp. Neurol. 436:42–51, 2001. © 2001 Wiley-Liss, Inc.

Mutations in theUnc5h3gene, a receptor for the netrin 1 ligand, result in abnormal migrations of both Purkinje and granule cells to regions outside the cerebellum and of granule cells to regions within the cerebellum. Because both Purkinje and granule cells express this molecule, we sought to determine whether one or both of these cell types are the primary target of the mutation.Chimeric mice were made between wild-type ROSA26 transgenic mouse embryos (whose cells express β-galactosidase) andUnc5h3mutant embryos. The resulting chimeric brains exhibited a range of phenotypes. Chimeras that had a limited expression of the extracerebellar phenotype (movement of cerebellar cells into the colliculus and midbrain tegmentum) and the intracerebellar phenotype (migration of granule cells into white matter) had a normal-appearing cerebellum, whereas chimeras that had more ectopic cells had attenuated anterior cerebellar lobules. Furthermore, the colonization of colliculus and midbrain tegmentum by cerebellar cells was not equivalent in all chimeras, suggesting different origins for extracerebellar ectopias in these regions.The granule cells of the extracerebellar ectopias were almost entirely derived fromUnc5h3/Unc5h3mutant embryos, whereas the ectopic Purkinje cells were a mixture of both mutant and wild-type cells. Intracerebellar ectopias in the chimera were composed exclusively of mutant granule cells. These findings demonstrate that both inside and outside the cerebellum, the granule cell is the key cell type to demarcate the boundaries of the cerebellum.

Dominantly inherited mutations in an endoplasmic reticulum protein called VAPB have been found in a subset of patients with a rare familial form of amyotrophic lateral sclerosis (ALS). In this issue, Tsuda et al. (2008) identify a secreted form of VAPB that binds directly to Eph receptors inducing their activation and signaling, providing fresh insights into ALS pathogenesis, including non-neuronal aspects of this disorder.