Search

Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disease that affects 1/350 individuals in the United Kingdom. The cause of amyotrophic lateral sclerosis is unknown in the majority of cases. Two-sample Mendelian randomization enables causal inference between an exposure, such as the serum concentration of a specific metabolite, and disease risk. We obtained genome-wide association study summary statistics for serum concentrations of 566 metabolites which were population matched with a genome-wide association study of amyotrophic lateral sclerosis. For each metabolite, we performed Mendelian randomization using an inverse variance weighted estimate for significance testing. After stringent Bonferroni multiple testing correction, our unbiased screen revealed three metabolites that were significantly linked to the risk of amyotrophic lateral sclerosis: Estrone-3-sulphate and bradykinin were protective, which is consistent with literature describing a male preponderance of amyotrophic lateral sclerosis and a preventive effect of angiotensin-converting enzyme inhibitors which inhibit the breakdown of bradykinin. Serum isoleucine was positively associated with amyotrophic lateral sclerosis risk. All three metabolites were supported by robust Mendelian randomization measures and sensitivity analyses; estrone-3-sulphate and isoleucine were confirmed in a validation amyotrophic lateral sclerosis genome-wide association study. Estrone-3-sulphate is metabolized to the more active estradiol by the enzyme 17β-hydroxysteroid dehydrogenase 1; further, Mendelian randomization demonstrated a protective effect of estradiol and rare variant analysis showed that missense variants within HSD17B1, the gene encoding 17β-hydroxysteroid dehydrogenase 1, modify risk for amyotrophic lateral sclerosis. Finally, in a zebrafish model of C9ORF72-amyotrophic lateral sclerosis, we present evidence that estradiol is neuroprotective. Isoleucine is metabolized via methylmalonyl-CoA mutase encoded by the gene MMUT in a reaction that consumes vitamin B12. Multivariable Mendelian randomization revealed that the toxic effect of isoleucine is dependent on the depletion of vitamin B12; consistent with this, rare variants which reduce the function of MMUT are protective against amyotrophic lateral sclerosis. We propose that amyotrophic lateral sclerosis patients and family members with high serum isoleucine levels should be offered supplementation with vitamin B12.

A hexanucleotide repeat expansion (HRE) within the chromosome 9 open reading frame 72 (C9orf72) gene is the most prevalent cause of amyotrophic lateral sclerosis/fronto-temporal dementia (ALS/FTD). Current evidence suggests HREs induce neurodegeneration through accumulation of RNA foci and/or dipeptide repeat proteins (DPR). C9orf72 patients are known to have transactive response DNA binding protein 43 kDa (TDP-43) proteinopathy, but whether there is further cross over between C9orf72 pathology and the pathology of other ALS sub-types has yet to be revealed. To address this, we generated and characterised two zebrafish lines expressing C9orf72 HREs. We also characterised pathology in human C9orf72-ALS cases. In addition, we utilised a reporter construct that expresses DsRed under the control of a heat shock promoter, to screen for potential therapeutic compounds. Both zebrafish lines showed accumulation of RNA foci and DPR. Our C9-ALS/FTD zebrafish model is the first to recapitulate the motor deficits, cognitive impairment, muscle atrophy, motor neuron loss and mortality in early adulthood observed in human C9orf72-ALS/FTD. Furthermore, we identified that in zebrafish, human cell lines and human post-mortem tissue, C9orf72 expansions activate the heat shock response (HSR). Additionally, HSR activation correlated with disease progression in our C9-ALS/FTD zebrafish model. Lastly, we identified that the compound ivermectin, as well as riluzole, reduced HSR activation in both C9-ALS/FTD and SOD1 zebrafish models. Thus, our C9-ALS/FTD zebrafish model is a stable transgenic model which recapitulates key features of human C9orf72-ALS/FTD, and represents a powerful drug-discovery tool. Electronic supplementary material The online version of this article (10.1186/s40478-018-0629-7) contains supplementary material, which is available to authorized users.

Multiple sclerosis (MS) is a chronic, immune-mediated demyelinating disease of the central nervous system. Animal models of MS have been critical for elucidating MS pathological mechanisms and how they may be targeted for therapeutic intervention. Here we review the most commonly used animal models of MS. Although these animal models cannot fully replicate the MS disease course, a number of models have been developed to recapitulate certain stages. Experimental autoimmune encephalomyelitis (EAE) has been used to explore neuroinflammatory mechanisms and toxin-induced demyelinating models to further our understanding of oligodendrocyte biology, demyelination and remyelination. Zebrafish models of MS are emerging as a useful research tool to validate potential therapeutic candidates due to their rapid development and amenability to genetic manipulation.

Summary Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways, including RNA processing, axonal transport, and protein homeostasis. We report ALS-causing mutations within the gene encoding the glycosyltransferase GLT8D1. Exome sequencing in an autosomal-dominant ALS pedigree identified p.R92C mutations in GLT8D1, which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site, and both R92C and G78W changes impair GLT8D1 enzyme activity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish consistent with ALS. Relative toxicity of mutations in model systems mirrors clinical severity. In conclusion, we have linked ALS pathophysiology to inherited mutations that diminish the activity of a glycosyltransferase enzyme., Graphical Abstract, Highlights • ALS-causing mutations found within the gene encoding the glycosyltransferase GLT8D1 • Five ALS-associated GLT8D1 mutations proximate to the substrate binding site • GLT8D1 mutations exhibit in vitro cytotoxicity and impair enzyme activity • GLT8D1 mutations induce motor deficits in zebrafish consistent with ALS, Amyotrophic lateral sclerosis (ALS) is an incurable neurodegeneration. Cooper-Knock et al. report ALS-causing mutations within GLT8D1. Mutations are associated with the substrate binding site and impair enzyme activity. Mutated GLT8D1 is neurotoxic and induces an ALS-like zebrafish phenotype.

Neurovascular coupling (through which local cerebral blood flow changes in response to neural activation are mediated) is impaired in many diseases including diabetes. Current preclinical rodent models of neurovascular coupling rely on invasive surgery and instrumentation, but transgenic zebrafish coupled with advances in imaging techniques allow non-invasive quantification of cerebrovascular anatomy, neural activation, and cerebral vessel haemodynamics. We therefore established a novel non-invasive, non-anaesthetised zebrafish larval model of neurovascular coupling, in which visual stimulus evokes neuronal activation in the optic tectum that is associated with a specific increase in red blood cell speed in tectal blood vessels. We applied this model to the examination of the effect of glucose exposure on cerebrovascular patterning and neurovascular coupling. We found that chronic exposure of zebrafish to glucose impaired tectal blood vessel patterning and neurovascular coupling. The nitric oxide donor sodium nitroprusside rescued all these adverse effects of glucose exposure on cerebrovascular patterning and function. Our results establish the first non-mammalian model of neurovascular coupling, offering the potential to perform more rapid genetic modifications and high-throughput screening than is currently possible using rodents. Furthermore, using this zebrafish model, we reveal a potential strategy to ameliorate the effects of hyperglycemia on cerebrovascular function.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways including RNA processing, axonal transport and protein homeostasis. Here we report mutations in a new ALS gene encoding the glycosyltransferase GLT8D1; this class of proteins has not previously been associated with neurodegeneration. Exome sequencing in an autosomal dominant ALS pedigree identified p.R92C mutations in GLT8D1 which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS-association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site and R92C reduces GLT8D1 enzyme activity ~2-fold. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish consistent with ALS. Relative toxicity of mutations in model systems mirrors clinical severity. In conclusion, we have discovered a previously uncharacterised pathophysiological pathway for ALS caused by inherited mutations within a glycosyltransferase enzyme.

Sequence of transgene injected to create C9-HRE transgenic zebrafish. (PDF 120 kb)

The transgenic SOD1G93A mouse is a model of human amyotrophic lateral sclerosis (ALS) and recapitulates many of the pathological hallmarks observed in humans, including motor neuron degeneration in the brain and the spinal cord. In mice, neurodegeneration particularly impacts on the facial nuclei in the brainstem. Motor neurons innervating the whisker pad muscles originate in the facial nucleus of the brain stem, with contractions of these muscles giving rise to “whisking” one of the fastest movements performed by mammals.\ud \ud A longitudinal study was conducted on SOD1G93A mice and wild-type litter mate controls, comparing: (i) whisker movements using high-speed video recordings and automated whisker tracking, and (ii) facial nucleus degeneration using MRI. Results indicate that while whisking still occurs in SOD1G93A mice and is relatively resistant to neurodegeneration, there are significant disruptions to certain whisking behaviours, which correlate with facial nuclei lesions, and may be as a result of specific facial muscle degeneration. We propose that measures of mouse whisker movement could potentially be used in tandem with measures of limb dysfunction as biomarkers of disease onset and progression in ALS mice and offers a novel method for testing the efficacy of novel therapeutic compounds.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways including RNA processing, axonal transport and protein homeostasis. Here we report mutations in a new ALS gene encoding the glycosyltransferase GLT8D1; this class of proteins has not previously been associated with neurodegeneration. Exome sequencing in an autosomal dominant ALS pedigree identified p.R92C mutations in GLT8D1 which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS-association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site and both R92C and G78W changes impair GLT8D1 enzyme activity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish consistent with ALS. Relative toxicity of mutations in model systems mirrors clinical severity. In conclusion, we have discovered a previously uncharacterised pathophysiological pathway for ALS caused by inherited mutations within a glycosyltransferase enzyme.

Neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are characterized by the presence of protein inclusions in the affected neurons. Emerging data indicate that protein misfolding may be of mechanistic importance in these diseases.1 Mutations in the ubiquitously expressed superoxide dismutase (SOD1) gene account for 20% of cases of the familial form of ALS. More than 150 mutations in the SOD1 gene have been discovered, including the point mutations G93R and G85R.2 Recent studies also implicate SOD1 in the sporadic form of ALS and suggest a prionlike propagation of misfolded SOD1.3–5 Interestingly, some of the newly identified genes implicated in ALS, such as TARDBP and FUS, are also proteins that show a high propensity to misfold and prionlike activity.6 However, we still do not know the precise mechanism by which mutant proteins cause toxicity.5,7 The emerging consensus view is that multiple interacting pathophysiological factors, including protein misfolding, contribute to the neuronal toxicity in ALS.8,9 Despite progress in revealing multiple molecular processes involved in disease pathology, relatively little is known about when and how the disekease, which starts focally, spreads throughout the motor network.10–12 Interestingly, even in the subtypes of ALS caused by SOD1 mutations, there is considerable phenotypic heterogeneity. Ravits and La Spada12 hypothesized that despite disease heterogeneity, the disease poses common themes that may involve common mechanisms. They propose that ALS may in fact be an orderly, actively propagating process and that fundamental molecular mechanisms may be uniform. The zebrafish is emerging as a useful tool for studying neurological diseases relevant to humans. Previously, we had shown that mutant sod1 transgenic fish show the hallmarks of adult onset neurodegenerative ALS, including defective motor performance, motor neuron loss, a loss of neuromuscular connectivity, and muscle atrophy.13 The aforementioned observations demonstrate the usefulness of the zebrafish as a model for this disease. However, among the current limitations when working with in vivo models of ALS is the lack of a good readout for the presymptomatic course of the disease. The zebrafish offer great advantages in studying early disease processes, as they develop rapidly, reaching postembryonic life at around 3 days postfertilization (dpf), which is developmentally similar to the neonatal mouse (for a comparison of developmental stages in human, mouse, and zebrafish, see Table 1). Moreover, the embryonic and larval zebrafish spinal cord is functionally and anatomically similar to that of humans, yet it is also optically transparent and experimentally accessible, making it ideal for the study of spinal circuits in normal and pathophysiological conditions.14 TABLE 1 Comparison of Neural Developmental Stages in Humans, Mice, and Zebrafish In the current study, we monitored in vivo early neurological changes caused by mutant sod1 gene. The sod1 zebrafish ALS model harbors a fluorescent heat shock stress response (HSR) reporter gene (hsp70-DsRed). The HSR is an endogenous cellular pathway that attempts to refold the damaged proteins in stressed cells, although this response is not always sufficient or beneficial.15 Thus, the HSR-mediated DsRed fluorescence in the sod1 zebrafish model of ALS represents a useful tool for monitoring perturbations in cellular homeostasis caused by sod1 mutation. This facilitates the mapping of disease focality and spread through the central nervous system (CNS) by the spatiotemporal readout of the neuronal stress response in the spinal cord of mutant zebrafish and provides an understanding of the cells and networks involved in disease propagation in ALS. We present evidence that the HSR is an indicator of early pathogenic processes occurring in neurons. The HSR is first observed at embryonic stages, in discrete populations of inhibitory interneurons in the spinal cord, and is followed by dysregulation of glycine release from these inhibitory interneurons. Furthermore, we observe that following interneuron dysfunction, motor neurons start exhibiting neuronal stress. More interestingly, we show that motor neurons showing the HSR also show dysfunctional neuromuscular junctions (NMJs). Taken together, our observations suggest that the mutant sod1-induced HSR is a robust predictor of neuronal dysfunction and thus is a reliable marker of disease pathogenesis. Finally, we also show that the neuronal stress readout can be used to identify neuroprotective compounds such as riluzole and identify biological targets that may ameliorate early pathophysiological disease processes that are currently not well explored. Although the sod1 zebrafish model may by itself not be sufficient in developing new therapies for ALS, this model system would provide a rapid way to triage compounds for screening in higher vertebrate models, with the potential for more rapid identification of promising compounds for translation into human clinical trials.

Table of SSMD β-value hit selection criteria. (DOC 32 kb)

Effects of drugs tested in human ALS in the mutant sod1 ZNSstress assay. (DOC 43 kb)

The zebrafish community has been steadily growing in the last 20 years in Europe. Given the federal structure of Europe, this increase in zebrafish research generated a need for a strategic forum to identify and discuss exciting new areas of research and funding opportunities as well as to address infrastructural and legal issues of experimentation, transport, and husbandry of zebrafish. To foster this exchange, the European Union (EU)-funded network EuFishBioMed (Cost Action BM0804) organized an international scientific meeting of zebrafish principal investigators in Padova, Italy, in March this year. More than 120 researchers from all over the globe presented their latest work in talks and posters. A number of workshops addressed future directions of research and infrastructural issues.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that, for ∼80% of patients, is fatal within five years of diagnosis. To better understand ALS, animal models have been essential; however, only rodent models of ALS exhibit the major hallmarks of the disease. Here, we report the generation of transgenic zebrafish overexpressing mutant Sod1. The construct used to generate these lines contained the zebrafish sod1 gene and ∼16 kb of flanking sequences. We generated lines expressing the G93R mutation, as well as lines expressing wild-type Sod1. Focusing on two G93R lines, we found that they displayed the major phenotypes of ALS. Changes at the neuromuscular junction were observed at larval and adult stages. In adulthood the G93R mutants exhibited decreased endurance in a swim tunnel test. An analysis of muscle revealed normal muscle force, however, at the end stage the fish exhibited motoneuron loss, muscle atrophy, paralysis and premature death. These phenotypes were more severe in lines expressing higher levels of mutant Sod1 and were absent in lines overexpressing wild-type Sod1. Thus, we have generated a vertebrate model of ALS to complement existing mammal models.

GLE1 mutations cause lethal congenital contracture syndrome 1 (LCCS1), a severe autosomal recessive fetal motor neuron disease, and more recently have been associated with amyotrophic lateral sclerosis (ALS). The gene encodes a highly conserved protein with an essential role in mRNA export. The mechanism linking Gle1 function to motor neuron degeneration in humans has not been elucidated, but increasing evidence implicates abnormal RNA processing as a key event in the pathogenesis of several motor neuron diseases. Homozygous gle1(-/-) mutant zebrafish display various aspects of LCCS, showing severe developmental abnormalities including motor neuron arborization defects and embryonic lethality. A previous gene expression study on spinal cord from LCCS fetuses indicated that oligodendrocyte dysfunction may be an important factor in LCCS. We therefore set out to investigate the development of myelinating glia in gle1(-/-) mutant zebrafish embryos. While expression of myelin basic protein (mbp) in hindbrain oligodendrocytes appeared relatively normal, our studies revealed a prominent defect in Schwann cell precursor proliferation and differentiation in the posterior lateral line nerve. Other genes mutated in LCCS have important roles in Schwann cell development, thereby suggesting that Schwann cell deficits may be a common factor in LCCS pathogenesis. These findings illustrate the potential importance of glial cells such as myelinating Schwann cells in motor neuron diseases linked to RNA processing defects.

The onset of amyotrophic lateral sclerosis (ALS) is conventionally considered as commencing with the recognition of clinical symptoms. We propose that, in common with other neurodegenerations, the pathogenic mechanisms culminating in ALS phenotypes begin much earlier in life. Animal models of genetically determined ALS exhibit pathological abnormalities long predating clinical deficits. The overt clinical ALS phenotype may develop when safety margins are exceeded subsequent to years of mitochondrial dysfunction, neuroinflammation or an imbalanced environment of excitation and inhibition in the neuropil. Somatic mutations, the epigenome and external environmental influences may interact to trigger a metabolic cascade that in the adult eventually exceeds functional threshold. A long preclinical and subsequent presymptomatic period pose a challenge for recognition, since it offers an opportunity for protective and perhaps even preventive therapeutic intervention to rescue dysfunctional neurons. We suggest, by analogy with other neurodegenerations and from SOD1 ALS mouse studies, that vulnerability might be induced in the perinatal period.

In the third millennium, competitive advantage in drug development will derive from expertise in two areas: 1) the ability to prioritize and triage hits from a combinatorial chemistry/high-throughput screening experiment and pursue only those hits most likely to succeed through clinical development, and 2) the ability to identify those patients capable of mounting a therapeutic response with minimal toxic effects. Pharmacogenomics, the branch of genomics addressing molecular pharmacology and toxicology, is anticipated to streamline drug development by addressing these issues. Pharmacogenomics includes two separate disciplines: expression pharmacogenomics and pharmacogenetics. Typically, they are regarded as unique fields and are pursued independently from each other. Here, we describe a pharmacogenomic strategy that combines and integrates both fields to create a single robust program. GeneCalling, a rapid, comprehensive differential transcript expression profiling technique, is applied to rodent models of drug response to identify novel markers predictive of drug efficacy and toxicity. SeqCalling, a high-throughput transcript sequencing strategy with a coding region bias, has identified 120,000 novel human single nucleotide polymorphisms (SNPs). Novel pharmacogenetic candidates are then identified by searching the human orthologs of rodent drug response genes for SeqCalling SNPs that can be pursued in systematic genotype screens to verify clinical correlations. In this manner, GeneCalling expression pharmacogenomics identifies markers capable of triaging leads from hits and SeqCalling converts a subset of these markers into pharmacogenetic correlates capable of identifying appropriately responsive patients. Drug Dev. Res. 49:54–64, 2000. © 2000 Wiley-Liss, Inc.

Corticotropin-releasing hormone (CRH) is widely recognized as the primary mediator of the neuroendocrine and behavioral responses to stress, including stress-induced anxiety. The biological activity of CRH and other mammalian CRH-like peptides, such as urocortin, may be modulated by CRH-binding protein (CRH-BP). To assess directly the CRH-BP function, we created a mouse model of CRH-BP deficiency by gene targeting. Basal adrenocorticotropic hormone and corticosterone levels are unchanged in the CRH-BP-deficient mice, and the animals demonstrate a normal increase in adrenocorticotropic hormone and corticosterone after restraint stress. In contrast, adult male CRH-BP-deficient mice show significantly reduced body weight when compared with wild-type controls. CRH-BP-deficient mice also exhibit a significant increase in anxiogenic-like behavior as assessed by the elevated plus maze and defensive withdrawal tests. The increased anorectic and anxiogenic-like behavior most likely is caused by increased “free” CRH and/or urocortin levels in the brain of CRH-BP-deficient animals, suggesting an important role for CRH-BP in maintaining appropriate levels of these peptides in the central nervous system.

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

Liposome-mediated intracellular delivery of clodronate is reported to selectively deplete mononuclear phagocytic cells such as macrophages that are important effector cells involved in the pathogenesis of neuropathies associated with demyelination and destruction of neuronal cells. Application of liposome-encapsulated clodronate (dichloromethylenediphosphonic acid disodium salt) is a method of choice to deplete macrophages to prevent such a neurodegeneration. In the present work, a comparison of an ion-exchange chromatography (IEC) and a capillary zone electrophoresis (CZE) method with indirect UV detection was performed and, based on the results of this comparison, a CZE assay was developed for quantitation of clodronate in mannosylated liposomes. This CZE method employed Nitroso-R salt (1-nitroso-2-naphthol-3,6-disulphonic acid disodium salt) as background electrolyte with UV detection of the analyte at 254 nm. The assay for the determination of clodronate in mannosylated liposomes after their solubilization in 10 mM Triton X-100 showed acceptable within-day precision (repeatability), day-to-day precision (reproducibility) and linearity in the target quantitation range of 0.5-10.0 mg ml(-1). The method reported here can be used as part of the quality control during the preparation of liposome-encapsulated clodronate as a drug formulation for macrophage-mediated diseases.

As new lead discovery technologies of high throughput screening and rational drug design have been incorporated into pharmaceutical and biotechnology drug discovery programs, researchers have focused on the applying these new technologies in diseases traditionally neglected by for-profit drug discovery efforts. This article reviews general trends in orphan disease lead discovery, identifies best practices of orphan market drug discovery and provides an overview of recent ALS lead discovery programs and drug development according to these metrics. Best practices in orphan market drug discovery embodied by programs like the NIH Anticonvulsant Screening Program include the (1) management of timelines and priorities, (2) engagement of for-profit partners, (3) creative application of technology, (4) collaboration, and (5) flexibility. Recent trends in ALS lead discovery have been shaped not only by the predominance of animal models of disease over in vitro models, but also by the successes and best practices of these earlier orphan market drug discovery programs. The ALS Treatment Initiative, the Johns Hopkins Center for ALS Research, the ALS Association, and the ALS Therapy Development Foundation have all initiated lead discovery programs in the past several years which seek to utilize existing experimental models of the disease and challenge assumptions about the linear nature of the lead discovery and development process. The compounds currently in clinical evaluation for ALS were identified as leads from a variety of sources, further reinforcing the transforming effect these new lead discovery programs have had on drug discovery and development in ALS. We conclude our review with an overview of the challenges and opportunities lead discovery in ALS currently faces, ultimately concluding that ALS lead discovery, and indeed orphan market drug discovery in general, would most benefit from more centralized lead discovery management, expanded national access to core facilities for lead discovery, and matrixed simultaneous screening of multiple compounds for multiple neglected diseases.

913 WE WRITE TO COMMENT BRIEFLY on the neurological literature on blood-derived neural stem cells, and to offer a case report regarding the safety of a simple procedure to introduce stem cells to the cerebrospinal fluid (CSF) compartment. For decades there has been evidence for cells of common neural and hematopoietic potential; this phenomenon was originally demonstrated by the presence of pluripotent cells resident in the adult brain that could form hematopoietic colonies in irradiated animals, as well as antigenic cross-reactivity between certain neural and hematopoietic cells (1–3). Recent studies have reproduced and confirmed the pleuripotency of neuro-hematopoietic cells, and showed that “blood can turn into brain” and vice-versa (4–6). Dr. W. Krivit at Harvard was the first to show that bone marrow transplantation (BMT) ameliorates some forms of central nervous system (CNS) genetic and metabolic disease (7), thought to be due to cellular margination and passage of the blood–brain barrier, which raised the intriguing idea of grafting blood cells directly to the human CNS. Indeed, administration of bone marrow-derived cells to the brain with neuronal engraftment has been demonstrated in animal models (8), and published reports now show blood stem cell differentiation along various neural lineages, including neurons and astrocytes. In an important clinical experiment conducted in Sweden, a group of patients received an intravenous (i.v.) label of bromodeoxyuridine (BrdU), which detects division of new cells. This work proved that neural progenitor cells exist in the brain of the adult human (9), thus generalizing an observation from primates and other animals that had generated much controversy, because it challenged a long-held tenet that brain cells could not be replenished or regenerate in situ. The precise source of neural progenitor cells is still a matter of debate and speculation, but the consensus is that they appear to be enriched around vascular tissues in the forebrain subventricular zone, the ependyma, and the dentate gyrus of the hippocampus. Given the close association between the vasculature and the sources of neural stem cells (10), one possibility is that neural stem cells arise in the adult human brain both from primordial brain stem cells and from migratory hematopoietic-derived cells. Considering the lack of effective drugs for amyotrophic lateral sclerosis (ALS) and accumulating evidence that primordial blood-derived cells can adopt a neuronal or glial fate, in 1999 we began exploring a blood stem cell selection protocol for application to ALS, in parallel with long-term plans for other experimental approaches. The objective was to bring to the clinic any promising drug or treatment protocol that had already been approved for human use. CD341 stem cell selection protocols are widely used in bone marrow transplants and are approved by the U.S. Food and Drug Administration (FDA), although clinical application for ALS is “off-label.” Immune ablation followed by BMT has already been performed in studies on multiple sclerosis and other primary CNS conditions; however, the mechanism we anticipated is one of cell grafting, CNS-specific differentiation, and local trophic effects, rather than the reconstitution of pharmacologically depleted immune cells. To demonstrate safety and generate preliminary data in support of dosing and method of deliver, we transplanted adult human marrow and fetal CD341 cells directly into the nonimmunosuppressed monkey spinal cord and intrathecal space. The monkeys underwent laminectomies, and xenogeneic human cells were introduced to the midline of the dorsal spinal cord at multiple sites using a Hamilton syringe and 30-gauge needle, without adverse behavioral or serological responses. The limited work available in the literature on cord blood transplants to superoxide dismutase (SOD1) mice also supported our ra

α-Fetoprotein (AFP) transcription is activated early in hepatogenesis, but is dramatically repressed within several weeks after birth. AFP regulation is governed by multiple elements including three enhancers termed EI, EII, and EIII. All three AFP enhancers continue to be active in the adult liver, where EI and EII exhibit high levels of activity in pericentral hepatocytes with a gradual reduction in activity in a pericentral-periportal direction. In contrast to these two enhancers, EIII activity is highly restricted to a layer of cells surrounding the central veins. To test models that could account for position-dependent EIII activity in the adult liver, we have analyzed transgenes in which AFP enhancers EII and EIII were linked together. Our results indicate that the activity of EIII is dominant over that of EII, indicating that EIII is a potent negative regulatory element in all hepatocytes except those encircling the central veins. We have localized this negative activity to a 340-bp fragment. This suggests that enhancer III may be involved in postnatal AFP repression.

Transcription of the mouse alpha-fetoprotein (AFP) gene, which is expressed at high levels in the visceral endoderm of the yolk sac and fetal liver and at low levels in the fetal gut, is regulated by three distinct upstream enhancer regions. To investigate the activities of these regions, each enhancer was individually linked to a heterologous human beta-globin promoter fused to the mouse class I H-2Dd structural gene. When tested in transgenic mice, the beta-globin promoter alone has minimal activity. We find that all three enhancers activate the beta-globin promoter in an AFP-like pattern; i.e., activity is detected in the yolk sac, fetal liver, and fetal gut. The enhancers remain active in the livers and guts of adult mice, consistent with previous studies showing that postnatal AFP repression is due not to the loss of enhancer activity but to a dominant repressor region. Enhancer III also functions in the brain. In addition, these studies reveal that the three enhancers exhibit different position-dependent activities in the adult liver. Enhancers I and II are most active in hepatocytes surrounding the central vein, with a gradual decrease in activity along the hepatic plates toward the portal triad. Enhancer III is active exclusively in hepatocytes surrounding the central vein. These data represent the first examples of individual control elements exhibiting positionally regulated activity in adult liver.

Objective: To study the role of TDP-43 in a zebrafish model of Amyotrophic Lateral Sclerosis. Background Amyotrophic lateral sclerosis is a devastating neurodegenerative condition. TDP-43 positive inclusions, have been reported as the hallmark pathology of ALS and FTLD-U. A major RNA processing function of TDP-43 is regulation of splicing. In mouse and drosophila models, TDP-43 has been shown to be important during early embryogenesis. TDP-43 has been observed to mis-localize from the nucleus to the cytoplasm of the surviving motor neurons in the postmortem brain and spinal cord tissues. Thus raising the possibility that the loss of nuclear function of TDP-43 might be responsible in the disease pathogenesis. Zebrafish is a robust vertebrate model, which we have used as a platform to study the loss of function effects of TDP-43. Design/Methods: We identified tardbp and tardbpl as zebrafish orthologues of TARDBP. We have used Antisense Morpholino Oligonucleotides (AMO) to transiently knock down tardbp and tardbpl. We generated tardbp mutant, Y220X, which results in a nonsense mediated decay of tardbp. We studied the motor neurons, axonal out growth, swimming behavior, survival and neuromuscular junction architecture, immuno blotting, immunohistochemistry and qRT-PCR to characterize and confirm our findings. Results: In a transient knockout of tardbp resulted in a phenotype similar to ALS. The stable homozygous tardbp Y220X mutant zebrafish shows significant reduction in weight and length at 6 months of age (p tardbp and tardbpl sequences we have discovered a novel alternative splicing event and a resultant novel splice variant of tardbpl. Conclusions: We have demonstrated that tardbp and tardbpl are essential for the normal development of the zebrafish motor neurons. Loss of tardbp results in a novel alternative splice isoform and up regulation of the same, thus providing an in vivo model of TDP-43 auto-regulation. Understanding this novel regulatory loop could play a key role in the successful generation of zebrafish models relating to ALS genes such as TDP-43 and FUS-1, which have important RNA processing functions. Supported by: CAH is funded by an MRC Clinical Research Fellowship from MRC Centre for Developmental Biomedical Genetics (CDBG). Disclosure: Dr. Hewamadduma has nothing to disclose. Dr. Grierson has nothing to disclose. Dr. Moens has nothing to disclose. Dr. Helde has nothing to disclose. Dr. Ingham has nothing to disclose. Dr. Ramesh has nothing to disclose. Dr. Shaw has received personal compensation for activities with Sanofi Aventis. Dr. Shaw has received research support from Trophos and Biogen Idec.

Amyotrophic lateral sclerosis/motor neuron disease (ALS/MND) is a devastating disease characterised by progressive motor neuron loss leading to paralysis and death. Despite the knowledge that mutations in the SOD1 gene cause ALS relatively little is known about when and how the disease, which starts focally, spreads throughout the motor network in the CNS to cause neuronal death. Aggregation of mutant SOD1 is one proposed key mechanism contributing to neuronal injury. Cells react to the presence of misfolded proteins by inducing the heatshock response (HSR) that trigger chaperones to refold the damaged proteins, though this response is not always sufficient. Nevertheless, the HSR provides a good readout of the underlying cellular stress. We hypothesised that mutant sod1 expression in vulnerable cell groups would induce the HSR, marking cellular stress. Using the hsp70-DsRed reporter of cellular stress in the mutant sod1 transgenic fish we show that the HSR to mutant sod1 provides a sensitive tool to identify neuronal stress well before the onset of clinical symptoms. We show that the neuronal stress occurs initially in the inhibitory interneurons in the early embryonic spinal cord followed by stress in the spinal motor neurons of symptomatic adults. Most importantly, the axons of stressed motor neurons show abnormal neuromuscular junction (NMJ) with significant reduction or absence of post-synaptic contact with the muscle causing denervation. We show that riluzole, the only drug approved in the treatment of MND, significantly reduces the HSR. We can now use this zebrafish model to identify potential disease modifying therapeutic agents.

Background Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2–3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well suited for the study of diseasepathophysiology and also for high throughput (HT) drug screening. The mutant sod1 zebrafish model of ALS mimics the hallmark features of ALS. Using a fluorescence based readout of neuronal stress, we developed a high throughput (HT) screen to identify neuroprotective compounds. Results Here we show that the zebrafish screen is a robust system that can be used to rapidly screen thousands ofcompounds and also demonstrate that riluzole is capable of reducing neuronal stress in this model system. The screen shows optimal quality control, maintaining a high sensitivity and specificity withoutcompromising throughput. Most importantly, we demonstrate that many compounds previously failed in human clinical trials, showed no stress reducing activity in the zebrafish assay. Conclusion We conclude that HT drug screening using a mutant sod1 zebrafish is a reliable model system which supplemented with secondary assays would be useful in identifying drugs with potential for neuroprotective efficacy in ALS. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0122-3) contains supplementary material, which is available to authorized users.