Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrP^C proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.     

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch‐evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA‐type glutamate receptor activation. Transection experiments suggest that the circuits required for touch‐evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA‐type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Knockdown of amyloid precursor protein in zebrafish causes defects in motor axon outgrowth

Amyloid precursor protein (APP) plays a pivotal role in Alzheimer's disease (AD) pathogenesis, but its normal physiological functions are less clear. Combined deletion of the APP and APP-like protein 2 (APLP2) genes in mice results in post-natal lethality, suggesting that APP performs an essential, if redundant, function during embryogenesis. We previously showed that injection of antisense morpholino to reduce APP levels in zebrafish embryos caused convergent-extension defects. Here we report that a reduction in APP levels causes defective axonal outgrowth of facial branchiomotor and spinal motor neurons, which involves disorganized axonal cytoskeletal elements. The defective outgrowth is caused in a cell-autonomous manner and both extracellular and intracellular domains of human APP are required to rescue the defective phenotype. Interestingly, wild-type human APP rescues the defective phenotype but APPswe mutation, which causes familial AD, does not. Our results show that the zebrafish model provides a powerful system to delineate APP functions in vivo and to study the biological effects of APP mutations.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

The zebrafish detour gene is essential for cranial but not spinal motor neuron induction.

The zebrafish detour (dtr) mutation generates a novel neuronal phenotype. In dtr mutants, most cranial motor neurons, especially the branchiomotor, are missing. However, spinal motor neurons are generated normally. The loss of cranial motor neurons is not due to aberrant hindbrain patterning, failure of neurogenesis, increased cell death or absence of hh expression. Furthermore, activation of the Hh pathway, which normally induces branchiomotor neurons, fails to induce motor neurons in the dtr hindbrain. Despite this, not all Hh-mediated regulation of hindbrain development is abolished since the regulation of a neural gene by Hh is intact in the dtr hindbrain. Finally, dtr can function cell autonomously to induce branchiomotor neurons. These results suggest that detour encodes a component of the Hh signaling pathway that is essential for the induction of motor neurons in the hindbrain but not in the spinal cord and that dtr function is required for the induction of only a subset of Hh-mediated events in the hindbrain.     

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish

Unlike mammals, fish motor function can recover within 6–8 weeks after spinal cord injury (SCI). The motor function of zebrafish is regulated by dual control; the upper motor neurons of the brainstem and motor neurons of the spinal cord. In this study, we aimed to investigate the framework behind the regeneration of upper motor neurons in adult zebrafish after SCI. In particular, we investigated the cell survival of axotomized upper motor neurons and its molecular machinery in zebrafish brain. As representative nuclei of upper motor neurons, we retrogradely labeled neurons in the nucleus of medial longitudinal fasciculus (NMLF) and the intermediate reticular formation (IMRF) using a tracer injected into the lesion site of the spinal cord. Four to eight neurons in each thin sections of the area of NMLF and IMRF were successfully traced at least 1–15 days after SCI. TUNEL staining and BrdU labeling assay revealed that there was no apoptosis or cell proliferation in the axotomized neurons of the brainstem at various time points after SCI. In contrast, axotomized neurons labeled with a neurotracer showed increased expression of anti-apoptotic factors, such as Bcl-2 and phospho-Akt (p-Akt), at 1–6 days after SCI. Such a rapid increase of Bcl-2 and p-Akt protein levels after SCI was quantitatively confirmed by western blot analysis. These data strongly indicate that upper motor neurons in the NMLF and IMRF can survive and regrow their axons into the spinal cord through the rapid activation of anti-apoptotic molecules after SCI. The regrowing axons from upper motor neurons reached the lesion site at 10–15 days and then crossed at 4–6 weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine “irregular” patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.     

Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord

Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co‐injection of phosphorylation‐mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 911–920, 2013     

GDP-bound Gαi2 regulates spinal motor neuron differentiation through interaction with GDE2

Gαi proteins play major roles in the developing and mature nervous system, ranging from the control of cellular proliferation to modulating synaptic plasticity. Although best known for transducing signals from activated seven transmembrane G-protein coupled receptors (GPCRs) when bound to GTP, key cellular functions for Gαi-GDP are beginning to emerge. Here, we show that Gαi2 is expressed in motor neuron progenitors that are differentiating to form postmitotic motor neurons in the developing spinal cord. Ablation of Gαi2 causes deficits in motor neuron generation but no changes in motor neuron progenitor patterning or specification, consistent with a function for Gαi2 in regulating motor neuron differentiation. We show that Gαi2 function is mediated in part by its interaction with GDE2, a known regulator of motor neuron differentiation, and that disruption of the GDE2/Gαi2 complex in vivo causes motor neuron deficits analogous to Gαi2 ablation. Gαi2 preferentially associates with GDE2 when bound to GDP, invoking GPCR-independent functions for Gαi2 in the control of spinal motor neuron differentiation.     

Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

Cell adhesion molecules belonging to the immunoglobulin superfamily (IgSF) control synaptic specificity through hetero- or homophilic interactions in different regions of the nervous system. In the developing spinal cord, monosynaptic connections of exquisite specificity form between proprioceptive sensory neurons and motor neurons, however, it is not known whether IgSF molecules participate in regulating this process. To determine whether IgSF molecules influence the establishment of synaptic specificity in sensory-motor circuits, we examined the expression of 157 IgSF genes in the developing dorsal root ganglion (DRG) and spinal cord by in situ hybridization assays. We find that many IgSF genes are expressed by sensory and motor neurons in the mouse developing DRG and spinal cord. For instance, Alcam, Mcam, and Ocam are expressed by a subset of motor neurons in the ventral spinal cord. Further analyses show that Ocam is expressed by obturator but not quadriceps motor neurons, suggesting that Ocam may regulate sensory-motor specificity in these sensory-motor reflex arcs. Electrophysiological analysis shows no obvious defects in synaptic specificity of monosynaptic sensory-motor connections involving obturator and quadriceps motor neurons in Ocam mutant mice. Since a subset of Ocam⁺ motor neurons also express Alcam, Alcam or other functionally redundant IgSF molecules may compensate for Ocam in controlling sensory-motor specificity. Taken together, these results reveal that IgSF molecules are broadly expressed by sensory and motor neurons during development, and that Ocam and other IgSF molecules may have redundant functions in controlling the specificity of sensory-motor circuits.     

Duplicated Gephyrin Genes Showing Distinct Tissue Distribution and Alternative Splicing Patterns Mediate Molybdenum Cofactor Biosynthesis,Glycine Receptor Clustering,and Escape Behavior in Zebrafish

Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABA_A receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish.     

A new transgenic reporter line reveals expression of protocadherin 9 at a cellular level within the zebrafish central nervous system

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.     

Infantile spinale Muskelatrophie: mehr als eine Motoneuronerkrankung?

Infantile spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the ventral horn of the spinal cord leading to weakness and muscle atrophy and occurs as a result of homozygous deletions or mutations in the survival motor neuron (SMN 1) gene. Loss of SMN 1 leads to a dramatic reduction in survival motor neuron (SMN) protein in the motor neurons of the spinal cord and of the brain stem. The SMA disease severity ranges from extremely severe to a relatively mild adult onset form of proximal muscle atrophy. More recently, clinical case reports in patients and studies in animal models provided evidence that severe SMN protein deficiency not only results in loss of motor neurons but also to additional organ manifestations. These include the peripheral, central and autonomic nervous system, development and function of the heart and the digestive tract and metabolic deficiencies. Therefore, to develop the most efficient therapeutic approach and also prevent further complications in patients that may arise with extended survival following therapeutic interventions, it is necessary to investigate in detail the specific damage to every system independently. The comparison of the defects in SMA mouse models will provide valuable insights; however, phenotypic differences between mice and men still remain.     

Neuronal function of Tbx20 conserved from nematodes to vertebrates

The Tbx20 orthologue, mab-9, is required for development of the Caenorhabditis elegans hindgut, whereas several vertebrate Tbx20 genes promote heart development. Here we show that Tbx20 orthologues also have a role in motor neuron development that is conserved between invertebrates and vertebrates. mab-9 mutants exhibit guidance defects in dorsally projecting axons from motor neurons located in the ventral nerve cord. Danio rerio (Zebrafish) tbx20 morphants show defects in the migration patterns of motor neuron soma of the facial and trigeminal motor neuron groups. Human TBX20 is expressed in motor neurons in the developing hindbrain of human embryos and we show that human TBX20 can substitute for zebrafish tbx20 in promoting cranial motor neuron migration. mab-9 is also partially able to rescue the zebrafish migration defect, whereas other vertebrate T-box genes cannot. Conversely we show that the human TBX20 T-box domain can rescue motor neuron defects in C. elegans. These data suggest the functional equivalence of Tbx20 orthologues in regulating the development of specific motor neuron groups. We also demonstrate the functional equivalence of human and C. elegans Tbx20 T-box domains for regulating male tail development in the nematode even though these genes play highly diverged roles in organogenesis.