Julia E. Dallman

Translocation of CaM kinase II to synaptic sites in vivo

The idea that calcium/calmodulin-dependent protein kinase II (CaMKII) is strategically localized to excitatory synapses to exert its important role in long-term potentiation and other forms of neuronal plasticity is supported by the binding of CaMKII to isolated postsynaptic densities (PSD) in biochemical assays and by the finding in cultured neurons that PSD clusters of green fluorescent protein (GFP)-tagged CaMKII form in response to glutamate application or direct electrical stimulation. The observation that CaMKII also forms large clusters in response to ischemic insults, however, questions the physiological relevance of such translocations. Here we show that in intact zebrafish, repeated sensory stimulation resulted in reproducible and reversible translocation of GFP-CaMKII to the PSD in an identified interneuron in a sensorimotor circuit.

Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss

Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs(∗)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183(∗)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss.

A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation.

Identification of conserved proteins that act to establish the neuronal phenotype has relied predominantly on structural homologies of the underlying genes. In the case of the repressor element 1 silencing transcription factor (REST), a central player in blocking the neuronal phenotype in vertebrate non-neural tissue, the invertebrate homolog is absent, raising the possibility that distinct strategies are used to establish the CNS of invertebrates. Using a yeast two-hybrid screen designed specifically to identify functional analogs of REST, we show that Drosophila melanogaster uses a strategy that is functionally similar to, but appears to have evolved independently of, REST. The gene at the center of the strategy in flies encodes the repressor Tramtrack88 (Ttk88), a protein with no discernable homology to REST but that nonetheless is able to interact with the same transcriptional partners. Ttk88 uses the REST corepressor Drosophila CoREST to coordinately regulate a set of genes encoding the same neuronal hallmarks that are regulated by REST in vertebrates. Our findings indicate that repression is an important mechanism for regulating neuronal phenotype across phyla and suggest that co-option of a similar corepressor complex occurred to restrict expression of genes critical for neuronal function to a compartmentalized nervous system.

Spontaneous activity regulates calcium-dependent K+ current expression in developing ascidian muscle

1 In embryonic ascidian muscle, outward K+ currents develop in two stages: the initial expression of a slowly activating, voltage‐gated K+ current (IKv) near the time of neurulation is followed about 6 h later by a rapidly activating calcium‐activated K+ current (IK(Ca)). During this 6 h interval, inward Ca2+ currents (ICa) appear and the inward rectifier (IK(IR)), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development. 2 By recording activity in cell‐attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when IK(IR) is downregulated and outward K+ currents are slow. Action potentials occur at a mean frequency of 13.9 min−1. 3 When activity is blocked by the transient application of the Ca2+ channel blocker Cd2+, IK(Ca) fails to develop. This disruption is specific for IK(Ca): IK(IR) and ICa develop normally in activity‐blocked cells. Application of Cd2+ either before or after the window of activity has no effect. 4 The reappearance of IK(IR) and the development of IK(Ca) and the mature form of ICa are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity. 5 These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of IK(Ca) depends on activity.

Co‐ordinated modulation of Ca2+ and K+ currents during ascidian muscle development.

1. The development of Ca2+ and K+ currents was studied in ascidian muscle cells at twelve embryonic stages from gastrulation to the mature cell, a period of 24 h. A high degree of co‐ordination occurs between the development of the inwardly rectifying K+ current (IK(IR)), which sets the resting potential, and Ca2+ and outward K+ currents, which determine action potential waveform. 2. At neurulation IK(IR), which had been present since fertilization, begins to decrease, reaching 12% of its previous density in 6 h. IK(IR) then immediately begins to increase again, reaching its previous density in another 6 h. 3. When IK(IR) begins to decrease, a high‐threshold inactivating Ca2+ current and a slowly activating voltage‐gated K+ current appear. 4. When IK(IR) returns to its previous density, two new currents appear: a sustained Ca2+ current with the same voltage dependence, but different conotoxin sensitivity than the inactivating Ca2+ current; and a Ca(2+)‐dependent K+ current, which activates 8‐10 times faster and at potentials 20‐30 mV more negative than the voltage‐dependent K+ current. 5. The transient downregulation of IK(IR) destabilizes the resting potential and causes spontaneous action potentials to occur. Because IK(IR) is absent when only a slowly activating high‐threshold outward K+ current is present, these action potentials are long in duration. 6. The return of IK(IR) and the appearance of the rapidly activating Ca(2+)‐dependent K+ current eventually terminate this activity. The action potentials of the mature cell occur only on stimulation, and are 10 times shorter in duration than those in the immature cell.

Synaptic Homeostasis in a Zebrafish Glial Glycine Transporter Mutant

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition.

Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1-2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes.

Mutations in RIT1 cause Noonan syndrome – additional functional evidence and expanding the clinical phenotype

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS‐mitogen activated protein kinase (RAS‐MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK‐ERK signaling compared to wild‐type, underscoring gain‐of‐function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS‐MAPK/MEK‐ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Crossing the blood-brain-barrier with transferrin conjugated carbon dots: A zebrafish model study.

Drug delivery to the central nervous system (CNS) in biological systems remains a major medical challenge due to the tight junctions between endothelial cells known as the blood-brain-barrier (BBB). Here we use a zebrafish model to explore the possibility of using transferrin-conjugated carbon dots (C-Dots) to ferry compounds across the BBB. C-Dots have previously been reported to inhibit protein fibrillation, and they are also used to deliver drugs for disease treatment. In terms of the potential medical application of C-Dots for the treatment of CNS diseases, one of the most formidable challenges is how to deliver them inside the CNS. To achieve this in this study, human transferrin was covalently conjugated to C-Dots. The conjugates were then injected into the vasculature of zebrafish to examine the possibility of crossing the BBB in vivo via transferrin receptor-mediated endocytosis. The experimental observations suggest that the transferrin-C-Dots can enter the CNS while C-Dots alone cannot.

Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle

1 Early in development, ascidian muscle cells generate spontaneous, long‐duration action potentials that are mediated by a high‐threshold, inactivating Ca2+ current. This spontaneous activity is required for appropriate physiological development. 2 Mature muscle cells generate brief action potentials only in response to motor neuron input. The mature action potential is mediated by a high‐threshold sustained Ca2+ current. 3 Action potentials recorded from these two stages were imposed as voltage‐clamp commands on cells of the same and different stages from which they were recorded. This strategy allowed us to study how immature and mature Ca2+ currents are optimized to their particular functions. 4 Total Ca2+ entry during an action potential did not change during development. The developmental increase in Ca2+ current density exactly compensated for decreased spike duration. This compensation was a function purely of Ca2+ current density, not of the transition from immature to mature Ca2+ current types. 5 In immature cells, Ca2+ entry was spread out over the entire waveform of spontaneous activity, including the interspike voltage trajectory. This almost continuous Ca2+ entry may be important in triggering Ca2+‐dependent developmental programmes, and is a function of the slightly more negative voltage dependence of the immature Ca2+ current. 6 In contrast, Ca2+ entry in mature cells was confined to the action potential itself, because of the slightly more positive voltage dependence of the mature Ca2+ current. This may be important in permitting rapid contraction‐relaxation cycles during larval swimming. 7 The inactivation of the immature Ca2+ current serves to limit the frequency and burst duration of spontaneous activity. The sustained kinetics of the mature Ca2+ current permit high‐frequency firing during larval swimming.