Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

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 SOD1G85R-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.

Emotional dysmetria after cerebellar-pontine stroke: a case report.

INTRODUCTION: Pseudobulbar affect, or emotional dysregulation, commonly occurs following stroke. However, it is frequently missed in cases involving the cerebellum, resulting in a lack of treatment, which can directly impact stroke rehabilitation. CASE PRESENTATION: A 63-year-old Caucasian female with no history of mood disorders presented with gait instability, dysarthria, and right sided hemiplegia, secondary to cerebellar and pontine ischemic stroke from a basilar occlusion. She underwent endovascular therapy and her deficits gradually improved. However during recovery she began to develop uncontrollable tearfulness while retaining insight that her emotional expression was contextually inappropriate. She was treated with a selective serotonin reuptake inhibitor with reported improvements in her emotional regulation at one year follow up. CONCLUSION: This case highlights cerebellar injury as a potential cause of poorly regulated emotions, or an emotional dysmetria. The recognition of this disorder in patients with cerebellar or pontine strokes is critical, as untreated pseudobulbar affect can impact future stroke rehabilitation.

Seizures are a druggable mechanistic link between TBI and subsequent tauopathy.

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.

Traumatic brain injury can result from direct head concussions, rapid head movements, or a blast wave generated by an explosion. Traumatic brain injury often causes seizures in the short term and is a risk factor for certain dementias, including Alzheimer's disease and chronic traumatic encephalopathy in the long term. A protein called Tau undergoes a series of chemical changes in these dementias that makes it accumulate, form toxic filaments and kill neurons. The toxic abnormal Tau proteins are initially found only in certain regions of the brain, but they spread as the disease progresses. Previous studies in Alzheimer's disease and other diseases where Tau proteins are abnormal suggest that Tau can spread between neighboring neurons and this can be promoted by neuron activity. However, scientists do not know whether similar mechanisms are at work following traumatic brain injury. Given that seizures are very common following traumatic brain injury, could they be partly responsible for promoting dementia? To investigate this, researchers need animal models in which they can measure neural activity associated with traumatic brain injury and observe the spread of abnormal Tau proteins. Alyenbaawi et al. engineered zebrafish so that their Tau proteins would be fluorescent, making it possible to track the accumulation of aggregated Tau protein in the brain. Next, they invented a simple way to perform traumatic brain injury on zebrafish larvae by using a syringe to produce a pressure wave. After this procedure, many of the fish exhibited features consistent with progression towards dementia, and seizure-like behaviors. The results showed that post-traumatic seizures are linked to the spread of aggregates of abnormal Tau following traumatic brain injury. Alyenbaawi et al. also found that anticonvulsant drugs can lower the levels of abnormal Tau proteins in neurons, preventing cell death, and could potentially ameliorate dementias associated with traumatic brain injury. These drugs are already being used to prevent post-traumatic epilepsy, but more research is needed to confirm whether they reduce the risk or severity of Tau-related neurodegeneration.

Tryptophan 32 mediates SOD1 toxicity in a in vivo motor neuron model of ALS and is a promising target for small molecule therapeutics.

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 (SOD1W32S) 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 UBQLN4D90A, EtOH, or tryptophan-deficient human SOD1W32S. 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.

Photoreceptor Progenitors Depend Upon Coordination of gdf6a, thrß, and tbx2b to Generate Precise Populations of Cone Photoreceptor Subtypes.

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 thrß. 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 thrß in gdf6a mutants by using a thrß morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrß activity via expression of a dominant negative thrß (dnthrß) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrß in gdf6a mutants, we revealed that disrupting thrß 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 thrß 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 thrß.

Reduced Abundance and Subverted Functions of Proteins in Prion-Like Diseases: Gained Functions Fascinate but Lost Functions Affect Aetiology.

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-ß 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, PrPC, 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.

Impacts of the retinal environment and photoreceptor type on functional regeneration.

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.

Rapid Recovery of Visual Function Associated with Blue Cone Ablation in Zebrafish.

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.

gdf6a is required for cone photoreceptor subtype differentiation and for the actions of tbx2b in determining rod versus cone photoreceptor fate.

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.

Growth differentiation factor 6 as a putative risk factor in neuromuscular degeneration.

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.

Longitudinal fluorescent observation of retinal degeneration and regeneration in zebrafish using fundus lens imaging.

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.

Regeneration of cone photoreceptors when cell ablation is primarily restricted to a particular cone subtype.

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.

Contribution of growth differentiation factor 6-dependent cell survival to early-onset retinal dystrophies.

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-ß (TGF-ß) 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-ß 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.