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Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2 . 3 , which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1 −/− zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease. Cerebral Cavernous Malformations (CCM) are often caused by mutations in CCM1/KRIT1. Here, Chapman et al. elegantly show that the CCM complex promotes apoptosis by regulating zinc homeostasis and storage via a conserved mechanism that likely generates the pathological defects observed in CCM.

SLC13A5 encodes a citrate transporter highly expressed in the brain and is important for regulating intra- and extracellular citrate levels. Mutations in this gene cause rare infantile epilepsy characterized by lifelong seizures, developmental delays, behavioral deficits, poor motor progression, and language impairments. SLC13A5 individuals respond poorly to treatment options; yet drug discovery programs are limited due to a paucity of animal models that phenocopy human symptoms. Here, we used CRISPR/Cas9 to create loss-of-function mutations in slc13a5a and slc13a5b , the zebrafish paralogs to human SLC13A5 . slc13a5 mutant larvae showed cognitive dysfunction and sleep disturbances, consistent with SLC13A5 individuals. These mutants also exhibited fewer neurons and a concomitant increase in apoptosis across the optic tectum, a region important for sensory processing. Further, slc13a5 mutants displayed hallmark features of epilepsy, including an imbalance in glutamatergic and GABAergic excitatory-inhibitory gene expression, increased fosab expression, disrupted neurometabolism, and neuronal hyperexcitation as measured in vivo by extracellular field recordings and live calcium imaging. Mechanistically, we tested the involvement of NMDA signaling and zinc chelation in slc13a5 mutant epilepsy-like phenotypes. Slc13a5 protein co-localizes with excitatory NMDA receptors in wild-type zebrafish and NMDA receptor expression is upregulated in the brain of slc13a5 mutant larvae. Additionally, low levels of zinc are found in the plasma membrane of slc13a5 mutants. NMDA receptor suppression and ZnCl 2 treatment in slc13a5 mutant larvae rescued neurometabolic and hyperexcitable calcium events, as well as behavioral defects. These data provide empirical evidence in support of the hypothesis that excess extracellular citrate over-chelates the zinc ions needed to regulate NMDA receptor function, leading to sustained channel opening and an exaggerated excitatory response that manifests as seizures. These data show the utility of slc13a5 mutant zebrafish for studying SLC13A5 epilepsy and open new avenues for drug discovery.

Zebrafish regenerate damaged myocardial tissue very effectively. Hence, insights into the molecular networks underlying zebrafish heart regeneration might help develop alternative strategies to restore human cardiac performance. While TGF-β signaling has been implicated in zebrafish cardiac regeneration, the role of its individual ligands remains unclear. Here, we report the opposing expression response during zebrafish heart regeneration of two genes, mstnb and inhbaa , which encode TGF-β family ligands. Using gain-of-function (GOF) and loss-of-function (LOF) approaches, we show that these ligands mediate inverse effects on cardiac regeneration and specifically on cardiomyocyte (CM) proliferation. Notably, we find that Inhbaa functions as a CM mitogen and that its overexpression leads to accelerated cardiac recovery and scar clearance after injury. In contrast, mstnb GOF and inhbaa LOF both lead to unresolved scarring after cardiac injury. We further show that Mstnb and Inhbaa inversely control Smad2 and Smad3 transcription factor activities through alternate Activin type 2 receptors. Zebrafish can regenerate damaged myocardial tissue but it is unclear how this is regulated. Here, the authors show that two TGF-β family members, Mstnb and Inhbaa, have opposite effects in regeneration, with mstnb overexpression or inhbaa loss-of-function causing cardiac scarring after injury.

The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.

The genetics of many congenital heart diseases (CHDs) can only unsatisfactorily be explained by known chromosomal or Mendelian syndromes. Here, we present sequencing data of a family with a potentially multigenic origin of CHD. Twelve of nineteen family members carry a familial mutation [NM_004329.2:c.1328 G > A (p.R443H)] which encodes a predicted deleterious variant of BMPR1A. This mutation co-segregates with a linkage region on chromosome 1 that associates with the emergence of severe CHDs including Ebstein’s anomaly, atrioventricular septal defect, and others. We show that the continuous overexpression of the zebrafish homologous mutation bmpr1aa p . R438H within endocardium causes a reduced AV valve area, a downregulation of Wnt/ß-catenin signalling at the AV canal, and growth of additional tissue mass in adult zebrafish hearts. This finding opens the possibility of testing genetic interactions between BMPR1A and other candidate genes within linkage region 1 which may provide a first step towards unravelling more complex genetic patterns in cardiovascular disease aetiology.

IntroductionThe adult mammalian heart is unable to regenerate damaged muscle tissue after myocardial infarction (MI). Instead, the damaged muscle is replaced by a non-contractile and functionally inert fibrotic scar, which disturbs the performance of the heart and causes arrhythmia (Steinhauser and Lee, 2011). Consequently MI is one of the leading causes of deaths worldwide (Mozaffarian et al., 2016). Several fields of research are trying to develop novel regenerative approaches to address this problem and are majorly focusing on the stimulation of cardiomyocyte proliferation in situ (Bersell et al., 2009), the engraftment of stem cell-derived cardiomyocytes into the injured hearts (Chong et al., 2014), or the in situ trans-differentiation of fibroblasts into functional cardiomyocytes (Qian et al., 2012).However, unlike mammals, lower vertebrates including zebrafish (Poss et al., 2002) can effectively regenerate the damaged cardiac tissue after multiple types of injury, such ventricular resection (Poss et al., 2002), cryoinjury (Chablais et al., 2011), or genetic cardiomyocyte ablation (Wang et al., 2011). Lineage tracing experiments have suggested that spared cardiomyocytes in the vicinity of the injured area undergo dedifferentiation and proliferation, thereby giving rise to new cardiomyocytes which replace the injured muscle (Jopling et al., 2010; Kikuchi et al., 2010). This makes zebrafish a suitable model organism to dissect the various molecular mechanisms underlying the process of cardiac regeneration, which would contribute in developing novel regenerative therapies.Several signaling pathways such as retinoic acid, interleukin 6 and epidermal growth factor (EGF) signaling have been reported to be involved in cardiac regeneration (Fang et al., 2013; Kikuchi, 2014; Kikuchi et al., 2011b; Marín-Juez et al., 2016; Wu et al., 2016). However, to date, only Neuregulin (Nrg) and its co-receptor ERBB2 have been reported to possess mitogenic activity by inducing cardiomyocyte proliferation in both healthy and injured hearts of the fish and mammals (Bersell et al., 2009; D’Uva et al., 2015; Gemberling et al., 2015; Liu et al., 2010). This makes Nrg a fundamental subject of ongoing research to assess its therapeutic potential for the treatment of human heart patients (Polizzotti et al., 2015).Notably, TGF-β signaling pathway has been implicated in various developmental (Ahuja et al., 2016; Azhar et al., 2009) and disease conditions (Pohlers et al., 2009; Siegel and Massagué, 2003), however the role of its various components in regulating cardiac regeneration still remains unclear. The vertebrate TGF-β/Activin subfamily (TGF-β subfamily) of ligands includes Activins (INHBA, INHBB), GDFs (Myostatin/GDF8, GDF11) and TGF-β (TGFB1, TGFB2 and TGFB3) which bind Activin type 2 receptors (ACVR2A, ACVR2B, TGFBR2), leading to the recruitment and activation of Activin type 1 receptors (ACVR1B, TGFBR1, ACVR1C). This process is followed by the phosphorylation of downstream effectors Smad2/3, which bind to Smad4 and translocate to the nucleus, thereby modulating the target gene expression (Massagué and Gomis, 2006; Massagué, 2012; Sartori et al., 2014).Enhanced TGF-β signaling, including the upregulation of Myostatin (MSTN) (Castillero et al., 2015), Inhibin betaA (INHBA) (Yndestad et al., 2004) and TGF-β (Li et al., 1997) has been observed post MI in the mammalian heart, which further stimulates hypertrophy, apoptosis and endothelial-mesenchymal transition (Euler-Taimor and Heger, 2006; Zeisberg et al., 2007).

Despite the availability of nearly 40 approved anti-seizure medications (ASMs), at least one-third of individuals with epilepsy remain refractory to treatment, and many experience life-limiting cognitive or psychiatric side effects. Using a machine learning-guided platform, we identified PDE4 as an underexplored anti-seizure target, which became further validated based on its enriched expression in seizure-relevant brain regions and its potential to modulate excitatory/inhibitory neuronal tone via cAMP signaling. The pan-PDE4 inhibitor crisaborole partially protected against hyperthermia-induced seizures and reduced spontaneous seizures in Scn1a+/- mice, while rolipram and roflumilast showed no efficacy at tolerable doses. SN-2000, a first-in-kind allosteric modulator of PDE4B, was rationally designed for isoform selectivity and brain penetration, and demonstrated versatile reduction of seizure activity across multiple zebrafish and rodent genetic and acquired epilepsy models, with efficacy comparable to standard-of-care ASMs. SN-2000 also demonstrated favorable behavioral outcomes, reducing post-ictal aggression and anxiety-like behaviors, and improving cognitive performance in both wild-type and epileptic mice. These effects were linked to paradoxical regulation of excitatory and neuronal activity in the cortex and thalamus of epileptic mice, respectively, as well as elevated cAMP signaling and downstream pCREB activation. Together, these findings support PDE4B inhibition as a disease-relevant mechanism in epilepsy, and position SN-2000 as a promising therapeutic candidate offering seizure control without the neuropsychiatric burden of existing ASMs and potential pro-cognitive properties. SN-2000, a novel allosteric PDE4 inhibitor, reduces seizure activity and shows psychiatric-neutral and pro-cognitive properties in preclinical models

SLC13A5 encodes a citrate transporter highly expressed in the brain important for regulating intra- and extracellular citrate levels. Mutations in this gene cause a rare infantile epilepsy characterized by lifelong seizures, developmental delays, behavioral deficits, poor motor progression, and language impairments. SLC13A5 individuals respond poorly to treatment options; yet drug discovery programs are limited due to a paucity of animal models that phenocopy human symptoms. Here, we used CRISPR/Cas9 to create loss-of-function mutations in slc13a5a and slc13a5b, the zebrafish paralogs to human SLC13A5. slc13a5 mutant larvae showed cognitive dysfunction and sleep disturbances, consistent with SLC13A5 individuals. These mutants also exhibited fewer neurons and a concomitant increase in apoptosis across the optic tectum, a region important for sensory processing. slc13a5 mutants displayed hallmark features of epilepsy, including an imbalance in glutamatergic and GABAergic excitatory-inhibitory gene expression, disrupted neurometabolism, and neuronal hyperexcitation as measured in vivo by extracellular field recordings and live calcium imaging. Mechanistically, we tested the involvement of NMDA signaling in slc13a5 mutant epilepsy-like phenotypes. Slc13a5 protein co-localizes with excitatory NMDA receptors in wild-type zebrafish and blocking NMDA receptors in slc13a5 mutant larvae rescued bioenergetics, hyperexcitable calcium events, and behavioral defects. These data provide empirical evidence in support of the hypothesis that excess extracellular citrate over-chelates the ions needed to regulate NMDA receptor function, leading to sustained channel opening and an exaggerated excitatory response that manifests as seizures. These data show the utility of slc13a5 mutant zebrafish for studying SLC13A5 epilepsy and open new avenues for drug discovery.Competing Interest StatementThe authors have declared no competing interest.

Intrinsic and extrinsic inhibition of axonal and neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after SC damage. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local neurogenesis impacts functional recovery is unknown. We uncovered dynamic expression of myostatin b (mstnb) in a niche of dorsal ependymal progenitors after complete SC transection in zebrafish. Genetic loss-of-function in mstnb impaired functional recovery, although glial and axonal bridging across the lesion were unaffected. Using a series of transgenic reporter lines, we quantified the numbers of stem, progenitor, and neuronal cells in the absence of mstnb. We found neural stem cell proliferation was reduced, while newborn neurons were increased in mstnb null tissues, suggesting mstnb is a negative regulator of neurogenesis. Molecularly, neuron differentiation genes were upregulated, while the neural stem cell maintenance gene fgf1b was downregulated in mstnb mutants. Finally, we show that human FGF1 treatment rescued neuronal gene expression in mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb in adult zebrafish, and establish the importance of local neurogenesis for functional SC repair. Competing Interest Statement The authors have declared no competing interest.

Objective: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), with epilepsy as a common comorbidity. Current medications only provide partial relief to ataxia and/or seizures, making new drugs needed. Here, we investigate the utility of zebrafish kcna1a-/- as a model of EA1 with epilepsy by characterizing its phenotype and comparing the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. Methods: We used CRISPR/Cas9 mutagenesis to introduce a mutation in the sixth segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. We also carried out real-time qPCRs to measure the transcript levels of brain hyperexcitability markers and bioenergetic profiling of kcna1a-/- larvae to evaluate their metabolic health. Carbamazepine efficacy was tested using behavioral assessments in kcna1a-/- zebrafish and seizure frequency in Kcna1-/- mice. Results: kcna1a-/- zebrafish showed uncoordinated movements and locomotor deficits. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation. Extracellular field recordings and upregulated fosab transcript levels showed hyperexcitability of the kcna1a-/- brain. Further, vglut2a and gad1b transcript levels were altered, indicative of neuronal excitatory/inhibitory imbalance in the kcna1a-/- brain. Metabolic health was also compromised in kcna1a-/- as seen by a significant reduction in measures of cellular respiration. Notably, carbamazepine reduced the impaired startle response in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to human efficacy compared to rodents. Significance: We conclude that zebrafish kcna1a-/- larvae show ataxia and epilepsy-related phenotypes and that they are responsive to carbamazepine treatment, consistent with EA1 patients. This study supports the notion that these zebrafish disease models can be useful for drug screening as well as studying the underlying disease biology. Competing Interest Statement The authors have declared no competing interest.