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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1 G85R -GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease., Competing Interests: Conflict of interest J.-P. J. and S. P. are the owners of a patent US 15/532,909 titled “TDP-43-binding polypeptides useful for the treatment of neurodegenerative diseases”. J.-P. J. is the chief scientific officer of Imstar Therapeutics. The 3H1 misfolded SOD1 antibody used in this manuscript is owned by the University of British Columbia and licensed by ProMIS Neurosciences. N. R. C. is the Chief Scientific Officer of ProMIS Neurosciences. S. S. P. and N. R. C. have received consultation compensation from ProMIS and possess ProMIS stock and stock options. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Seizures and other forms of neurovolatility are emerging as druggable prodromal mechanisms that link traumatic brain injury (TBI) to the progression of later dementias. TBI neurotrauma has both acute and long-term impacts on health, and TBI is a leading risk factor for dementias, including chronic traumatic encephalopathy and Alzheimer's disease. Treatment of TBI already considers acute management of posttraumatic seizures and epilepsy, and impressive efforts have optimized regimens of antiepileptic drugs (AEDs) toward that goal. Here we consider that expanding these management strategies could determine which AED regimens best prevent dementia progression in TBI patients. Challenges with this prophylactic strategy include the potential consequences of prolonged AED treatment and that a large subset of patients are refractory to available AEDs. Addressing these challenges is warranted because the management of seizure activity following TBI offers a rare opportunity to prevent the onset or progression of devastating dementias.

Purpose: NRL is an influential transcription factor and central to animal modeling in ophthalmology. Disrupting NRL abrogates rod development and produces an excess of S-cones (also known as "UV cones" or "short-wavelength-sensitive1 [SWS1] cones"). Strikingly, mutations in zebrafish tbx2b produce the exact opposite phenotypes (excess rods and loss of SWS1 cones). We sought to define what genetic relationship exists, if any, between these transcription factors. We also infer whether these two phenotypes (altered rod abundance and altered SWS1 cone abundance) are independent versus inter-related., Methods: Zebrafish mutants were bred to disrupt nrl and tbx2b in concert. Rods and SWS1 cones were quantified and characterized at ultrastructural and transcriptional levels., Results: Considering single mutant zebrafish, we confirmed previously established phenotypes and noted that the number of rods lost in nrl-/- mutants is reflected by a concomitant increase in SWS1 cone abundance. The tbx2b-/- mutants present the opposite phenotype(s) but exhibit a similar trade-off in cell abundances, with lots of rods and a concomitant decrease in SWS1 cones. Double mutant nrl-/-;tbx2b-/- zebrafish recapitulate the nrl-/- mutant phenotype(s)., Conclusions: The tbx2b is thought to be required for producing SWS1 cones in zebrafish, but this can be over-ridden when nrl is absent. Regarding the altered cell abundances observed in either tbx2b-/- or nrl-/- mutants, the alterations in rod and SWS1 cones appear to not be two separate phenotypes but are instead a single intertwined outcome. The tbx2b and nrl are in an epistatic relationship, with nrl phenotypes dominating, implying that tbx2b is upstream of nrl in photoreceptor cell fate determination.

We describe a straightforward, scalable method for administering traumatic brain injury (TBI) to zebrafish larvae. The pathological outcomes appear generalizable for all TBI types, but perhaps most closely model closed-skull, diffuse lesion (blast injury) neurotrauma. The injury is delivered by dropping a weight onto the plunger of a fluid-filled syringe containing zebrafish larvae. This model is easy to implement, cost-effective, and provides a high-throughput system that induces brain injury in many larvae at once. Unique to vertebrate TBI models, this method can be used to deliver TBI without anesthetic or other metabolic agents. The methods simulate the main aspects of traumatic brain injury in humans, providing a preclinical model to study the consequences of this prevalent injury type and a way to explore early interventions that may ameliorate subsequent neurodegeneration. We also describe a convenient method for executing pressure measurements to calibrate and validate this method. When used in concert with the genetic tools readily available in zebrafish, this model of traumatic brain injury offers opportunities to examine many mechanisms and outcomes induced by traumatic brain injury. For example, genetically encoded fluorescent reporters have been implemented with this system to measure protein misfolding and neural activity via optogenetics., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.

Background: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multi-organ condition occurring with a 1 in 3800 prevalence in Alberta. This genetic disorder leads to vascular malformations in different organs including the lungs and brain, commonly affecting pulmonary vasculature leading to pulmonary arteriovenous malformations (PAVMs). PAVMs lead to right-to-left shunts, which may be associated with neurologic complications. We aimed to evaluate and summarize the reported neurologic manifestations of individuals with HHT with pre-existing PAVMs., Methods: We performed a qualitative systematic review to determine available literature on neurological complications among patients with PAVMs and HHT. Published studies included observational studies, case studies, prospective studies, and cohort studies including search terms HHT, PAVMs, and various neurologic complications using MEDLINE and EMBASE., Results: A total of 449 manuscripts were extracted including some duplicates of titles, abstracts, and text which were screened. Following this, 23 publications were identified for inclusion in the analysis. Most were case reports ( n = 15). PAVMs were addressed in all these articles in association with various neurological conditions ranging from cerebral abscess, ischemic stroke, hemorrhagic stroke, embolic stroke, and migraines., Conclusion: Although HHT patients with PAVMs are at risk for a variety of neurological complications compared to those without PAVMs, the quality and volume of evidence characterizing this association is low. Individuals with PAVMs have a high prevalence of neurological manifestations such as cerebral abscess, transient ischemic attack, cerebral embolism, hemorrhage, and stroke. Mitigating stroke risk by implementing proper standardized screening techniques for PAVMs is invaluable in preventing increased mortality.

Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252., Competing Interests: H.K. holds the position of Editorial Board Member for Function and is blinded from reviewing or making decisions for the manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general., (© 2022. The Author(s).)

Photoreceptor dysfunctions and degenerative diseases are significant causes of vision loss in patients, with few effective treatments available. Targeted interventions to prevent or reverse photoreceptor-related vision loss are not possible without a thorough understanding of the underlying mechanism leading to disease, which is exceedingly difficult to accomplish in the human system. Cone diseases are particularly challenging to model, as some popular genetically modifiable model animals are nocturnal with a rod-dominant visual system and cones that have dissimilarities to human cones. As a result, cone diseases, which affect visual acuity, colour perception, and central vision in patients, are generally poorly understood in terms of pathology and mechanism. Zebrafish (Danio rerio) provide the opportunity to model photoreceptor diseases in a diurnal vertebrate with a cone-rich retina which develops many macular degeneration-like pathologies. Zebrafish undergo external development, allowing early-onset retinal diseases to be detected and studied, and many ophthalmic tools are available for zebrafish visual assessment during development and adulthood. There are numerous zebrafish models of photoreceptor disease, spanning the various types of photoreceptor disease (developmental, rod, cone, and mixed photoreceptor diseases) and genetic/molecular cause. In this review, we explore the features of zebrafish that make them uniquely poised to model cone diseases, summarize the established zebrafish models of inherited photoreceptor disease, and discuss how disease in these models compares to the human presentation, where applicable. Further, we highlight the contributions of these zebrafish models to our understanding of photoreceptor biology and disease, and discuss future directions for utilising and investigating these diverse models., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The vertebrate eye is a vital sensory organ that has long fascinated scientists, but the details of how this organ evolved are still unclear. The vertebrate eye is distinct from the simple photoreceptive organs of other non-vertebrate chordates and there are no clear transitional forms of the eye in the fossil record. To investigate the evolution of the eye we can examine the eyes of the most ancient extant vertebrates, the hagfish and lamprey. These jawless vertebrates are in an ideal phylogenetic position to study the origin of the vertebrate eye but data on eye/retina development in these organisms is limited. New genomic and gene expression data from hagfish and lamprey suggest they have many of the same genes for eye development and retinal neurogenesis as jawed vertebrates, but functional work to determine if these genes operate in retinogenesis similarly to other vertebrates is missing. In addition, hagfish express a marker of proliferative retinal cells ( Pax6 ) near the margin of the retina, and adult retinal growth is apparent in some species. This finding of eye growth late into hagfish ontogeny is unexpected given the degenerate eye phenotype. Further studies dissecting retinal neurogenesis in jawless vertebrates would allow for comparison of the mechanisms of retinal development between cyclostome and gnathostome eyes and provide insight into the evolutionary origins of the vertebrate eye., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bradshaw and Allison.)

Cellular Prion Protein (PrP C ) is a well-studied protein as the substrate for various progressive untreatable neurodegenerative diseases. Normal functions of PrP C are poorly understood, though recent proteomic and transcriptomic approaches have begun to reveal common themes. We use our compound prp1 and prp2 knockout mutant zebrafish at three days post fertilization to take a transcriptomic approach to investigating potentially conserved PrP C functions during development. Gene ontology analysis shows the biological processes with the largest changes in gene expression include redox processing, transport and cell adhesion. Within these categories several different gene families were prevalent including the solute carrier proteins, cytochrome p450 enzymes and protocadherins. Continuing from previous studies identifying cell adhesion as an important function of PrP C we found that in addition to the protocadherins there was a significant reduction in transcript abundance of both ncam1a and st8sia2 . These two genes are involved in the early development of vertebrates. The alterations in cell adhesion transcripts were consistent with past findings in zebrafish and mouse prion protein mutants; however E-cadherin processing after prion protein knockdown failed to reveal any differences compared with wild type in either our double prp1/prp2 mutant fish or after prp1 morpholino knockdown. Our data supports a cross species conserved role for PrP C in the development and maintenance of the central nervous system, particularly by regulating various and important cell adhesion processes.

The ancient paralogs premelanosome protein ( PMEL ) and glycoprotein nonmetastatic melanoma protein B ( GPNMB ) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130 , including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.

In light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and Δ 9 -tetrahydrocannabinol (THC) until the end of gastrulation (1-10 h post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented Calcium-Modulated Photoactivatable Ratiometric Integrator (CaMPARI) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and their receptors to understand the implications of cannabis consumption on fetal development.

Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and can also be activated by some Group 9/10 transition metals, which is believed to mediate immune hypersensitivity reactions. In this work, we test whether TLR4 can be activated by the Group 10 metal platinum and the platinum-based chemotherapeutic cisplatin. Cisplatin is invaluable in childhood cancer treatment but its use is limited due to a permanent hearing loss (cisplatin-induced ototoxicity, CIO) adverse effect. We demonstrate that platinum and cisplatin activate pathways downstream of TLR4 to a similar extent as the known TLR4 agonists LPS and nickel. We further show that TLR4 is required for cisplatin-induced inflammatory, oxidative, and cell death responses in hair cells in vitro and for hair cell damage in vivo. Finally, we identify a TLR4 small molecule inhibitor able to curtail cisplatin toxicity in vitro. Thus, our findings indicate that TLR4 is a promising therapeutic target to mitigate CIO., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Traumatic brain injury (TBI) is a prominent risk factor for dementias including tauopathies like chronic traumatic encephalopathy (CTE). The mechanisms that promote prion-like spreading of Tau aggregates after TBI are not fully understood, in part due to lack of tractable animal models. Here, we test the putative role of seizures in promoting the spread of tauopathy. We introduce 'tauopathy reporter' zebrafish expressing a genetically encoded fluorescent Tau biosensor that reliably reports accumulation of human Tau species when seeded via intraventricular brain injections. Subjecting zebrafish larvae to a novel TBI paradigm produced various TBI features including cell death, post-traumatic seizures, and Tau inclusions. Bath application of dynamin inhibitors or anticonvulsant drugs rescued TBI-induced tauopathy and cell death. These data suggest a role for seizure activity in the prion-like seeding and spreading of tauopathy following TBI. Further work is warranted regarding anti-convulsants that dampen post-traumatic seizures as a route to moderating subsequent tauopathy., Competing Interests: HA, RK, LL, RK, MD, QB, HW, EB, WA No competing interests declared

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6 + ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.

Zebrafish are an instrumental system for the generation of photoreceptor degeneration models, which can be utilized to determine underlying causes of photoreceptor dysfunction and death, and for the analysis of potential therapeutic compounds, as well as the characterization of regenerative responses. We review the wealth of information from existing zebrafish models of photoreceptor disease, specifically as they relate to currently accepted taxonomic classes of human rod and cone disease. We also highlight that rich, detailed information can be derived from studying photoreceptor development, structure, and function, including behavioural assessments and in vivo imaging of zebrafish. Zebrafish models are available for a diversity of photoreceptor diseases, including cone dystrophies, which are challenging to recapitulate in nocturnal mammalian systems. Newly discovered models of photoreceptor disease and drusenoid deposit formation may not only provide important insights into pathogenesis of disease, but also potential therapeutic approaches. Zebrafish have already shown their use in providing pre-clinical data prior to testing genetic therapies in clinical trials, such as antisense oligonucleotide therapy for Usher syndrome., Competing Interests: The authors declare no conflict of interest.

The transcription factor NRL (neural retina leucine zipper) has been canonized as the master regulator of photoreceptor cell fate in the retina. NRL is necessary and sufficient to specify rod cell fate and to preclude cone cell fate in mice. By engineering zebrafish, we tested if NRL function has conserved roles beyond mammals or beyond nocturnal species, i.e., in a vertebrate possessing a greater and more typical diversity of cone sub-types. Transgenic expression of Nrl from zebrafish or mouse was sufficient to induce rod photoreceptor cells. Zebrafish nrl -/- mutants lacked rods (and had excess UV-sensitive cones) as young larvae; thus, the conservation of Nrl function between mice and zebrafish appears sound. Strikingly, however, rods were abundant in adult nrl -/- null mutant zebrafish. Rods developed in adults despite Nrl protein being undetectable. Therefore, a yet-to-be-revealed non-canonical pathway independent of Nrl is able to specify the fate of some rod photoreceptors., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

The accumulation of tau protein in the form of filamentous aggregates is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). These dementias share traumatic brain injury (TBI) as a prominent risk factor. Tau aggregates can transfer between cells and tissues in a "prion-like" manner, where they initiate the templated misfolding of normal tau molecules. This enables the spread of tau pathology to distinct parts of the brain. The evidence that tauopathies spread via prion-like mechanisms is considerable, but work detailing the mechanisms of spread has mostly used in vitro platforms that cannot fully reveal the tissue-level vectors or etiology of progression. We review these issues and then briefly use TBI and CTE as a case study to illustrate aspects of tauopathy that warrant further attention in vivo. These include seizures and sleep/wake disturbances, emphasizing the urgent need for improved animal models. Dissecting these mechanisms of tauopathy progression continues to provide fresh inspiration for the design of diagnostic and therapeutic approaches.

Photoreceptor disease results in irreparable vision loss and blindness, which has a dramatic impact on quality of life. Pathogenic mutations in RP1L1 lead to photoreceptor degenerations such as occult macular dystrophy and retinitis pigmentosa. RP1L1 is a component of the photoreceptor axoneme, the backbone structure of the photoreceptor's light-sensing outer segment. We generated an rp1l1 zebrafish mutant using CRISPR/Cas9 genome editing. Mutant animals had progressive photoreceptor functional defects as determined by electrophysiological assessment. Optical coherence tomography showed gaps in the photoreceptor layer, disrupted photoreceptor mosaics, and thinner retinas. Mutant retinas had disorganized photoreceptor outer segments and lipid-rich subretinal drusenoid deposits between the photoreceptors and retinal pigment epithelium. Our mutant is a novel model of RP1L1 -associated photoreceptor disease and the first zebrafish model of photoreceptor degeneration with reported subretinal drusenoid deposits, a feature of age-related macular degeneration.

The vertebrate jaw is a versatile feeding apparatus. To function, it requires a joint between the upper and lower jaws, so jaw joint defects are often highly disruptive and difficult to study. To describe the consequences of jaw joint dysfunction, we engineered two independent null alleles of a single jaw joint marker gene, nkx3.2 , in zebrafish. These mutations caused zebrafish to become functionally jawless via fusion of the upper and lower jaw cartilages (ankylosis). Despite lacking jaw joints, nkx3.2 mutants survived to adulthood and accommodated this defect by: (a) having a remodeled skull with a fixed open gape, reduced snout and enlarged branchial region; and (b) performing ram feeding in the absence of jaw-generated suction. The late onset and broad extent of phenotypic changes in the mutants suggest that modifications to the skull are induced by functional agnathia, secondarily to nkx3.2 loss of function. Interestingly, nkx3.2 mutants superficially resemble ancient jawless vertebrates (anaspids and furcacaudiid thelodonts) in overall head shape. Because no homology exists in individual skull elements between these taxa, the adult nkx3.2 phenotype is not a reversal but rather a convergence due to similar functional requirements of feeding without moveable jaws. This remarkable analogy strongly suggests that jaw movements themselves dramatically influence the development of jawed vertebrate skulls. Thus, these mutants provide a unique model with which to: (a) investigate adaptive responses to perturbation in skeletal development; (b) re-evaluate evolutionarily inspired interpretations of phenocopies generated by gene knockdowns and knockouts; and (c) gain insight into feeding mechanics of the extinct agnathans., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Objective: Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model., Methods: In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca 2+ imaging with the ratiometric Ca 2+ sensor CaMPARI., Results: Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca 2+ imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior., Significance: This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes., (© 2020 International League Against Epilepsy.)

Genetically encodable calcium ion (Ca 2+ ) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca 2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

Disrupted sleep is a major feature of Alzheimer's disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (Aβ) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aβ affects sleep are unknown. We demonstrate in zebrafish that Aβ acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aβ oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aβ forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signaling cascade. Our data indicate that Aβ can trigger a bi-directional sleep/wake switch. Alterations to the brain's Aβ oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signaling events., Competing Interests: GÖ, SL, PL, WA, JR No competing interests declared, (© 2020, Özcan et al.)

It has been proposed that Amyloid β Precursor Protein (APP) might act as a rheostat controlling neuronal excitability, but mechanisms have remained untested. APP and its catabolite Aβ are known to impact upon synapse function and dysfunction via their interaction with the prion protein (PrP C ), suggesting a candidate pathway. Here we test if PrP C is required for this APP function in vivo, perhaps via modulating mGluR5 ion channels. We engineered zebrafish to lack homologs of PrP C and APP, allowing us to assess their purported genetic and physiological interactions in CNS development. We generated four appa null alleles as well as prp1 -/- ;appa -/- double mutants (engineering of prp1 mutant alleles is described elsewhere). Unexpectedly, appa -/- and compound prp1 -/- ;appa -/- mutants are viable and lacked overt phenotypes (except being slightly smaller than wildtype fish at some developmental stages). Zebrafish prp1 -/- mutants were substantially more sensitive to appa knockdown than wildtype fish, and both zebrafish prp1 and mammalian Prnp mRNA were significantly able to partially rescue this effect. Further, appa -/- mutants exhibited increased seizures upon exposure to low doses of convulsant. The mechanism of this seizure susceptibility requires prp1 insomuch that seizures were significantly dampened to wildtype levels in prp1 -/- ;appa -/- mutants. Inhibiting mGluR5 channels, which may be downstream of PrP C , increased seizure intensity only in prp1 -/- mutants, and this seizure mechanism required intact appa. Taken together, these results support an intriguing genetic interaction between prp1 and appa with their shared roles impacting upon neuron hyperexcitability, thus complementing and extending past works detailing their biochemical interaction(s)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endocannabinoids (eCBs) mediate their effects through actions on several receptors, including the cannabinoid receptors CB 1 R and CB 2 R. The role played by eCBs in the development of locomotor systems is not fully understood. In this study, we investigated the roles of the eCB system in zebrafish development by pharmacologically inhibiting CB 1 R and CB 2 R (with AM251 and AM630, respectively) in either the first or second day of development. We examined the morphology of motor neurons and we determined neuromuscular outputs by quantifying the amount of swimming in 5 days post-fertilization larvae. Blocking CB 2 R during the first day of development resulted in gross morphological deficits and reductions in heart rate that were greater than those following treatment with the CB 1 R blocker AM251. Blocking CB 1 Rs from 0 to 24 h post-fertilization resulted in an increase in the number of secondary and tertiary branches of primary motor neurons, whereas blocking CB 2 Rs had the opposite effect. Both treatments manifested in reduced levels of swimming. Additionally, blocking CB 1 Rs resulted in greater instances of non-inflated and partially inflated swim bladders compared with AM630 treatment, suggesting that at least some of the deficits in locomotion may result from an inability to adjust buoyancy. Together, these findings indicate that the eCB system is pivotal to the development of the locomotor system in zebrafish, and that perturbations of the eCB system early in life may have detrimental effects., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Pigmentary glaucoma (PG) is a common glaucoma subtype that results from release of pigment from the iris, called pigment dispersion syndrome (PDS), and its deposition throughout the anterior chamber of the eye. Although PG has a substantial heritable component, no causative genes have yet been identified. We used whole exome sequencing of two independent pedigrees to identify two premelanosome protein (PMEL) variants associated with heritable PDS/PG. PMEL encodes a key component of the melanosome, the organelle essential for melanin synthesis, storage and transport. Targeted screening of PMEL in three independent cohorts (n = 394) identified seven additional PDS/PG-associated non-synonymous variants. Five of the nine variants exhibited defective processing of the PMEL protein. In addition, analysis of PDS/PG-associated PMEL variants expressed in HeLa cells revealed structural changes to pseudomelanosomes indicating altered amyloid fibril formation in five of the nine variants. Introduction of 11-base pair deletions to the homologous pmela in zebrafish by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method caused profound pigmentation defects and enlarged anterior segments in the eye, further supporting PMEL's role in ocular pigmentation and function. Taken together, these data support a model in which missense PMEL variants represent dominant negative mutations that impair the ability of PMEL to form functional amyloid fibrils. While PMEL mutations have previously been shown to cause pigmentation and ocular defects in animals, this research is the first report of mutations in PMEL causing human disease., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

SOD1 misfolding, toxic gain of function, and spread are proposed as a pathological basis of amyotrophic lateral sclerosis (ALS), but the nature of SOD1 toxicity has been difficult to elucidate. Uniquely in SOD1 proteins from humans and other primates, and rarely in other species, a tryptophan residue at position 32 (W32) is predicted to be solvent exposed and to participate in SOD1 misfolding. We hypothesized that W32 is influential in SOD1 acquiring toxicity, as it is known to be important in template-directed misfolding. We tested if W32 contributes to SOD1 cytotoxicity and if it is an appropriate drug target to ameliorate ALS-like neuromuscular deficits in a zebrafish model of motor neuron axon morphology and function (swimming). Embryos injected with human SOD1 variant with W32 substituted for a serine (SOD1 W32S ) had reduced motor neuron axonopathy and motor deficits compared to those injected with wildtype or disease-associated SOD1. A library of FDA-approved small molecules was ranked with virtual screening based on predicted binding to W32, and subsequently filtered for analogues using a pharmacophore model based on molecular features of the uracil moiety of a small molecule previously predicted to interact with W32 (5'-fluorouridine or 5'-FUrd). Along with testing 5'-FUrd and uridine, a lead candidate from this list was selected based on its lower toxicity and improved blood brain barrier penetrance; telbivudine significantly rescued SOD1 toxicity in a dose-dependent manner. The mechanisms whereby the small molecules ameliorated motor neuron phenotypes were specifically mediated through human SOD1 and its residue W32, because these therapeutics had no measurable impact on the effects of UBQLN4 D90A , EtOH, or tryptophan-deficient human SOD1 W32S . By substituting W32 for a more evolutionarily conserved residue (serine), we confirmed the significant influence of W32 on human SOD1 toxicity to motor neuron morphology and function; further, we performed pharmaceutical targeting of the W32 residue for rescuing SOD1 toxicity. This unique residue offers future novel insights into SOD1 stability and toxic gain of function, and therefore poses an potential target for drug therapy., (Copyright © 2018. Published by Elsevier Inc.)

Purpose: Replacing cone photoreceptors, the units of the retina necessary for daytime vision, depends upon the successful production of a full variety of new cones from, for example, stem cells. Using genetic experiments in a model organism with high cone diversity, zebrafish, we map the intersecting effects of cone development factors gdf6a, tbx2b, and thrβ., Methods: We investigated these genes of interest by using genetic combinations of mutants, gene knockdown, and dominant negative gene expression, and then quantified cone subtype outcomes (which normally develop in tightly regulated ratios)., Results: Gdf6a mutants have reduced blue cones and, discovered here, reduced red cones. In combined gdf6a/tbx2b disruption, the loss of gdf6a in heterozygous tbx2b mutants reduced UV cones. Intriguingly, when we disrupted thrβ in gdf6a mutants by using a thrβ morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrβ activity via expression of a dominant negative thrβ (dnthrβ) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrβ in gdf6a mutants, we revealed that disrupting thrβ activity did not change gdf6a mutant cone phenotypes., Conclusions: Gdf6a loss directly affects blue and red cones and indirectly affects UV cones by increasing sensitivity to additional disruption, such as reduced tbx2b, resulting in fewer UV cones. The effects of thrβ change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. The effects of gdf6a on UV, blue, and red cone development overlap with, but likely supersede, those of thrβ.

Normally folded prion protein is abundant in the CNS and remarkably conserved, suggesting that it has important functions, yet these functions have remained elusive. Now the work of Parrie et al. has codified a requirement for prion protein in adult neurogenesis. Their insightful use of prion protein knockout and over-expressing mice, combined with the well-characterized olfactory system site of neurogenesis, demonstrated that prion protein promotes proliferation and survival of adult neurons. The work provides a unique independent confirmation of prion protein playing a role in neuroprotection, especially extending the conclusion beyond models using acute injury. Parrie et al. (2018) further show that prion protein is required for CNS axon guidance. A growing list of phenotypes associated with prion protein loss are coincident with symptoms of neurodegenerative disease and dementia, though it remains contentious whether any such disruption of prion protein function contributes to disease aetiology. Perhaps most intriguingly, identifying the developmental functions for prion protein opens new avenues to understand the evolution of prion protein: what history led to a CNS protein that is conserved and abundant paradoxically being both dispensable for life and the template for devastating disease?, (Copyright © 2018 Elsevier Inc. All rights reserved.)

Normally folded prion protein (PrP C ) and its functions in healthy brains remain underappreciated compared with the intense study of its misfolded forms ("prions," PrP Sc ) during the pathobiology of prion diseases. This impedes the development of therapeutic strategies in Alzheimer's and prion diseases. Disrupting the zebrafish homologs of PrP C has provided novel insights; however, mutagenesis of the zebrafish paralog prp2 did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the prp1 paralog. Here, we generated zebrafish prp1 loss-of-function mutant alleles and dual prp1 -/- ;prp2 -/- mutants. Zebrafish prp1 -/- and dual prp1 -/- ;prp2 -/- mutants resemble mammalian Prnp knockouts insofar as they lack overt phenotypes, which surprisingly contrasts with reports of severe developmental phenotypes when either prp1 or prp2 is knocked down acutely. Previous studies suggest that PrP C participates in neural cell development/adhesion, including in zebrafish where loss of prp2 affects adhesion and deposition patterns of lateral line neuromasts. In contrast with the expectation that prp1 's functions would be redundant to prp2 , they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of prp1 blunted the seizure susceptibility phenotypes observed in prp2 mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In summary, prion mutant fish lack the overt phenotypes previously predicted, and instead they have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The connectivity amongst photoreceptors is critical to their function, as it underpins lateral inhibition and effective translation of stimuli into neural signals. Despite much work characterizing second-order interneurons in the outer retina, the synapses directly connecting photoreceptors have often been overlooked. Telodendria are fine processes that connect photoreceptor pedicles. They have been observed in diverse vertebrate groups, yet their roles in vision remain speculative. Here, we visualize telodendria via fluorescent protein expression in photoreceptor subtypes. We characterized short wavelength cone telodendria in adult and larval zebrafish retina. Additionally, in the larval retina, we investigated rod telodendria and UV cone telodendria in mutant and transgenic retinas with altered complements of cone types. In the adult retina, telodendria are twice as abundant and branch almost twice as often on blue cones compared to UV cones. Pedicles of neighboring UV and blue cones typically converge into contiguous pairs, despite the regular spacing of their cell bodies. In contrast to adults, larval UV cone telodendria are more numerous (1.3 times) than blue cone telodendria. UV cone telodendria are not detectably affected by ablation of blue cones, and are reduced twofold in mutant larval retina with few UV cones. We thus saw no evidence that telodendria increase in number in the absence of their typical cellular neighbors. We also found that larval rod telodendria are less abundant than short wavelength cone telodendria. In summary, we describe the development and morphology of zebrafish photoreceptor synaptic connectivity toward appreciating the function of telodendria in visual signal processing., (© 2017 Wiley Periodicals, Inc.)

The misfolding of cellular prion protein (PrP C ) to form PrP Scrapie (PrP Sc ) is an exemplar of toxic gain-of-function mechanisms inducing propagated protein misfolding and progressive devastating neurodegeneration. Despite this, PrP C function in the brain is also reduced and subverted during prion disease progression; thus understanding the normal function of PrP C in healthy brains is key. Disrupting PrP C in mice has led to a myriad of controversial functions that sometimes map onto disease symptoms, including a proposed role in memory or learning. Intriguingly, PrP C interaction with amyloid beta (Aβ) oligomers at synapses has also linked its function to Alzheimer's disease and dementia in recent years. We set out to test the involvement of PrP C in memory using a disparate animal model, the zebrafish. Here we document an age-dependent memory decline in prp2 -/- zebrafish, pointing to a conserved and ancient role of PrP C in memory. Specifically, we found that aged (3-year-old) prp2 -/- fish performed poorly in an object recognition task relative to age-matched prp2 +/+ fish or 1-year-old prp2 -/- fish. Further, using a novel object approach (NOA) test, we found that aged (3-year-old) prp2 -/- fish approached the novel object more than either age-matched prp2 +/+ fish or 1-year-old prp2 -/- fish, but did not have decreased anxiety when we tested them in a novel tank diving test. Taken together, the results of the NOA and novel tank diving tests suggest an altered cognitive appraisal of the novel object in the 3-year-old prp2 -/- fish. The learning paradigm established here enables a path forward to study PrP C interactions of relevance to Alzheimer's disease and prion diseases, and to screen for candidate therapeutics for these diseases. The findings underpin a need to consider the relative contributions of loss- versus gain-of-function of PrP C during Alzheimer's and prion diseases, and have implications upon the prospects of several promising therapeutic strategies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Prions have served as pathfinders that reveal many aspects of proteostasis in neurons. The recent realization that several prominent neurodegenerative diseases spread via a prion-like mechanism illuminates new possibilities for diagnostics and therapeutics. Thus, key proteins in Alzheimer Disease and Amyotrophic lateral sclerosis (ALS), including amyloid-β precursor protein, Tau and superoxide dismutase 1 (SOD1), spread to adjacent cells in their misfolded aggregated forms and exhibit template-directed misfolding to induce further misfolding, disruptions to proteostasis and toxicity. Here we invert this comparison to ask what these prion-like diseases can teach us about the broad prion disease class, especially regarding the loss of these key proteins' function(s) as they misfold and aggregate. We also consider whether functional amyloids might reveal a role for subverted protein function in neurodegenerative disease. Our synthesis identifies SOD1 as an exemplar of protein functions being lost during prion-like protein misfolding, because SOD1 is inherently unstable and loses function in its misfolded disease-associated form. This has under-appreciated parallels amongst the canonical prion diseases, wherein the normally folded prion protein, PrP C , is reduced in abundance in fatal familial insomnia patients and during the preclinical phase in animal models, apparently via proteostatic mechanisms. Thus while template-directed misfolding and infectious properties represent gain-of-function that fascinates proteostasis researchers and defines (is required for) the prion(-like) diseases, loss and subversion of the functions attributed to hallmark proteins in neurodegenerative disease needs to be integrated into design towards effective therapeutics. We propose experiments to uniquely test these ideas., Competing Interests: The authors declare no conflict of interest.

Retinal regeneration is a promising central nervous system (CNS) target amongst the various stem cell therapy pursuits, due to its accessibility for manipulation and its disposition towards longitudinal monitoring of treatment safety and efficacy. We offer our perspective on current hurdles towards functional regeneration of cone photoreceptors. Cones are key: For patients suffering vision loss, cone photoreceptors are a required cellular component to restoring daytime vision, colour vision, and high acuity vision. The challenges of regenerating cones contrast with logistic challenges of regenerating rod photoreceptors, which underlines the importance of evaluating context in degeneration and regeneration studies. Foundational research is required to delineate the factors required to generate a diversity of cones in the human macula, and to coax both remaining and newly regenerating cones to rewire towards restoring daytime colour vision. A complex interplay between cell-intrinsic factors and the retinal environment determine both the specification of cone fates and the synaptic plasticity enabling their functional integration. Recent revelations that cellular materials are transferred amongst photoreceptor progenitors further emphasize the critical role of neighbouring cells in directing stem cell fates. From our vantage point, translation of stem cell therapies to restore the cone-rich human macula must be borne upon foundational research in cone-rich retinas. Research frameworks centered on patient outcomes should prioritize animal models and functional outputs that enable and report functional restoration of cone-mediated vision., Competing Interests: Conflicts of interest: None declared.

Hurdles in the treatment of retinal degeneration include managing the functional rewiring of surviving photoreceptors and integration of any newly added cells into the remaining second-order retinal neurons. Zebrafish are the premier genetic model for such questions, and we present two new transgenic lines allowing us to contrast vision loss and recovery following conditional ablation of specific cone types: UV or blue cones. The ablation of each cone type proved to be thorough (killing 80% of cells in each intended cone class), specific, and cell-autonomous. We assessed the loss and recovery of vision in larvae via the optomotor behavioural response (OMR). This visually mediated behaviour decreased to about 5% or 20% of control levels following ablation of UV or blue cones, respectively (P<0.05). We further assessed ocular photoreception by measuring the effects of UV light on body pigmentation, and observed that photoreceptor deficits and recovery occurred (p<0.01) with a timeline coincident to the OMR results. This corroborated and extended previous conclusions that UV cones are required photoreceptors for modulating body pigmentation, addressing assumptions that were unavoidable in previous experiments. Functional vision recovery following UV cone ablation was robust, as measured by both assays, returning to control levels within four days. In contrast, robust functional recovery following blue cone ablation was unexpectedly rapid, returning to normal levels within 24 hours after ablation. Ablation of cones led to increased proliferation in the retina, though the rapid recovery of vision following blue cone ablation was demonstrated to not be mediated by blue cone regeneration. Thus rapid visual recovery occurs following ablation of some, but not all, cone subtypes, suggesting an opportunity to contrast and dissect the sources and mechanisms of outer retinal recovery during cone photoreceptor death and regeneration., Competing Interests: The authors have declared that no competing interests exist.

Prion disease research has contributed much toward understanding other neurodegenerative diseases, including recent demonstrations that Alzheimer's disease (AD) and other neurodegenerative diseases are prion-like. Prion-like diseases involve the spread of degeneration between individuals and/or among cells or tissues via template directed misfolding, wherein misfolded protein conformers propagate disease by causing normal proteins to misfold. Here we use the premise that AD, amyotrophic lateral sclerosis, Huntington's disease, and other similar diseases are prion-like and ask: Can we apply knowledge gained from studies of these prion-like diseases to resolve debates about classical prion diseases? We focus on controversies about what role(s) protein loss-of-function might have in prion diseases because this has therapeutic implications, including for AD. We examine which loss-of-function events are recognizable in prion-like diseases by considering the normal functions of the proteins before their misfolding and aggregation. We then delineate scenarios wherein gain-of-function and/or loss-of-function would be necessary or sufficient for neurodegeneration. We consider roles of PrPC loss-of-function in prion diseases and in AD, and conclude that the conventional wisdom that prion diseases are 'toxic gain-of-function diseases' has limitations. While prion diseases certainly have required gain-of-function components, we propose that disease phenotypes are predominantly caused by deficits in the normal physiology of PrPC and its interaction partners as PrPC converts to PrPSc. In this model, gain-of-function serves mainly to spread disease, and loss-of-function directly mediates neuron dysfunction. We propose experiments and predictions to assess our conclusion. Further study on the normal physiological roles of these key proteins is warranted.

The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

Vertebrate ancestors had only cone-like photoreceptors. The duplex retina evolved in jawless vertebrates with the advent of highly photosensitive rod-like photoreceptors. Despite cones being the arbiters of high-resolution color vision, rods emerged as the dominant photoreceptor in mammals during a nocturnal phase early in their evolution. We investigated the evolutionary and developmental origins of rods in two divergent vertebrate retinas. In mice, we discovered genetic and epigenetic vestiges of short-wavelength cones in developing rods, and cell-lineage tracing validated the genesis of rods from S cones. Curiously, rods did not derive from S cones in zebrafish. Our study illuminates several questions regarding the evolution of duplex retina and supports the hypothesis that, in mammals, the S-cone lineage was recruited via the Maf-family transcription factor NRL to augment rod photoreceptors. We propose that this developmental mechanism allowed the adaptive exploitation of scotopic niches during the nocturnal bottleneck early in mammalian evolution., (Published by Elsevier Inc.)

Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2-/- mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.

Many neurons receive synapses in stereotypic proportions from converging but functionally distinct afferents. However, developmental mechanisms regulating synaptic convergence are not well understood. Here we describe a heterotypic mechanism by which one afferent controls synaptogenesis of another afferent, but not vice versa. Like other CNS circuits, zebrafish retinal H3 horizontal cells (HC) undergo an initial period of remodelling, establishing synapses with ultraviolet and blue cones while eliminating red and green cone contacts. As development progresses, the HCs selectively synapse with ultraviolet cones to generate a 5:1 ultraviolet-to-blue cone synapse ratio. Blue cone synaptogenesis increases in mutants lacking ultraviolet cones, and when transmitter release or visual stimulation of ultraviolet cones is perturbed. Connectivity is unaltered when blue cone transmission is suppressed. Moreover, there is no cell-autonomous regulation of cone synaptogenesis by neurotransmission. Thus, biased connectivity in this circuit is established by an unusual activity-dependent, unidirectional control of synaptogenesis exerted by the dominant input.

Functional vision restoration is within reach via stem cell therapy, but one of the largest obstacles is the derivation of colour-sensitive cone photoreceptors that are required for high-acuity daytime vision. To enhance progress made using nocturnal murine models, we instead utilize cone-rich zebrafish and herein investigate relationships between gdf6a and tbx2b in cone photoreceptor development. Growth/differentiation factor 6a (gdf6a), a bone morphogenetic protein family ligand, is an emerging factor in photoreceptor degenerative diseases. The T-box transcription factor tbx2b is required to specify UV cone photoreceptor fate instead of rod photoreceptor fate. Interactions between these factors in cone development would be unanticipated, considering the discrete phenotypes in their respective mutants. However, gdf6a positively modulates the abundance of tbx2b transcript during early eye morphogenesis, and we extended this conclusion to later stages of retinal development comprising the times when photoreceptors differentiate. Despite this, gdf6a-/- larvae possess a normal relative number of UV cones and instead present with a low abundance of blue cone photoreceptors, approximately half that of siblings (p<0.001), supporting a differential role for gdf6a amongst the spectral subtypes of cone photoreceptors. Further, gdf6a-/- larvae from breeding of compound heterozygous gdf6a+/-;tbx2b+/- mutants exhibit the recessive lots-of-rods phenotype (which also shows a paucity of UV cones) at significantly elevated rates (44% or 48% for each of two tbx2b alleles, χ2 p≤0.007 for each compared to expected Mendelian 25%). Thus the gdf6a-/- background sensitizes fish such that the recessive lots-of-rods phenotype can appear in heterozygous tbx2b+/- fish. Overall, this work establishes a novel link between tbx2b and gdf6a in determining photoreceptor fates, defining the nexus of an intricate pathway influencing the abundance of cone spectral subtypes and specifying rod vs. cone photoreceptors. Understanding this interaction is a necessary step in the refinement of stem cell-based restoration of daytime vision in humans.

Mutation of Glass bottom boat, the Drosophila homologue of the bone morphogenetic protein or growth/differentiation factor (BMP/GDF) family of genes in vertebrates, has been shown to disrupt development of neuromuscular junctions (NMJ). Here we tested whether this same conclusion can be broadened to vertebrate BMP/GDF genes. This analysis was also extended to consider whether such genes are required for NMJ maintenance in post-larval stages, as this would argue that BMP genes are viable candidates for analysis in progressive neuromuscular disease. Zebrafish mutants harboring homozygous null mutations in the BMP-family gene gdf6a were raised to adulthood and assessed for neuromuscular deficits. Fish lacking gdf6a exhibited decreased endurance (∼ 50%, p = 0.005) compared to wild type, and this deficit progressively worsened with age. These fish also presented with significantly disrupted NMJ morphology (p = 0.009), and a lower abundance of spinal motor neurons (∼ 50%, p<0.001) compared to wild type. Noting the similarity of these symptoms to those of Amyotrophic Lateral Sclerosis (ALS) model mice and fish, we asked if mutations in gdf6a would enhance the phenotypes observed in the latter, i.e. in zebrafish over-expressing mutant Superoxide Dismutase 1 (SOD1). Amongst younger adult fish only bigenic fish harboring both the SOD1 transgene and gdf6a mutations, but not siblings with other combinations of these gene modifications, displayed significantly reduced endurance (75%, p<0.05) and strength/power (75%, p<0.05), as well as disrupted NMJ morphology (p<0.001) compared to wild type siblings. Bigenic fish also had lower survival rates compared to other genotypes. Thus conclusions regarding a role for BMP ligands in effecting NMJ can be extended to vertebrates, supporting conservation of mechanisms relevant to neuromuscular degenerative diseases. These conclusions synergize with past findings to argue for further analysis of GDF6 and other BMP genes as modifier loci, potentially affecting susceptibility to ALS and perhaps a broader suite of neurodegenerative diseases.

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

The function of the cellular prion protein (PrP(C)) in healthy brains remains poorly understood, in part because Prnp knockout mice are viable. On the other hand, transient knockdown of Prnp homologs in zebrafish (including two paralogs, prp1 and prp2) has suggested that PrP(C) is required for CNS development, cell adhesion, and neuroprotection. It has been argued that zebrafish Prp2 is most similar to mammalian PrP(C), yet it has remained intransigent to the most thorough confirmations of reagent specificity during knockdown. Thus we investigated the role of prp2 using targeted gene disruption via zinc finger nucleases. Prp2(-/-) zebrafish were viable and did not display overt developmental phenotypes. Back-crossing female prp2(-/-) fish ruled out a role for maternal mRNA contributions. Prp2(-/-) larvae were found to have increased seizure-like behavior following exposure to the convulsant pentylenetetrazol (PTZ), as compared to wild type fish. In situ recordings from intact hindbrains demonstrated that prp2 regulates closing of N-Methyl-d-aspartate (NMDA) receptors, concomitant with neuroprotection during glutamate excitotoxicity. Overall, the knockout of Prp2 function in zebrafish independently confirmed hypothesized roles for PrP, identifying deeply conserved functions in post-developmental regulation of neuron excitability that are consequential to the etiology of prion and Alzheimer diseases., (Copyright © 2013. Published by Elsevier Inc.)

Purpose: Longitudinal observation of retinal degeneration and regeneration in animal models is time-consuming and expensive. To address this challenge, we used a custom fundus lens and zebrafish transgenic lines with cell-specific fluorescent reporters to document the state of individual retinal neurons in vivo., Methods: We empirically tested several versions of a custom fundus lens and assessed its capabilities under a stereomicroscope to image retinal neurons in transgenic zebrafish lines expressing fluorescent reporters. Vascular branch points provided spatial references enabling determination of whether changes induced by ablating photoreceptors were repaired over the course of several days., Results: Individual ultraviolet- and blue-sensitive cone photoreceptors were readily visualized in vivo, and green fluorescent protein-labeled blood vessels were used as landmarks to facilitate orientation. Sequential imaging of the same retinal areas over several weeks permitted documentation of photoreceptor reappearance in individual animals. Photoreceptor regeneration in these regions was evidenced by the reappearance of individual fluorescent cells., Conclusions: This technique permits real-time in vivo serial examination of individual fish, permitting temporal analysis of changes to the retinal mosaic. The key benefits this technique offers include that the same retinal locations can be recovered and viewed at multiple time points, that in vivo observations are comparable to those made ex vivo, and that fewer animals need to be euthanized over the course of an experiment. Our results promise the ability to detect individual cells, including reappearing cone photoreceptors, and to monitor disease progression during screening of therapies in an adult animal model of late onset disease.

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.

The four-eyed fish, Anableps anableps, has eyes with unusual morphological adaptations for simultaneous vision above and below water. The retina, for example, is divided such that one region receives light from the aerial field and the other from the aquatic field. To understand better the adaptive value of this partitioned retina, we characterized photoreceptor distribution using in situ hybridization. Cones expressing sws1, sws2b and rh2-2 (i.e. UV, and short wavelength-sensitive) opsins were found throughout the retina, whereas cones expressing rh2-1 (middle wavelength-sensitive) were largely limited to the ventral retina and those expressing lws (long wavelength-sensitive) opsins were only expressed in the dorsal retina. We next asked when this pattern evolved relative to the 'four-eyed' morphology. We characterized opsin expression in Jenynsia onca, a member of the sister genus to Anableps with typical teleost eye morphology. In J. onca, sws1, sws2b, rh2-2 and rh2-1 opsins were expressed throughout the retina; while lws opsins were not expressed in the ventral retina. Thus, the change that coincides with the evolution of unusual anablepid eye morphology is the loss of rh2-1 expression in the dorsal retina, probably to accommodate increased lws opsin expression. The retinal area that samples aerial light appears not to have changed with respect to photoreceptor transcription.

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.