Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential offtarget sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.

Loss of function mutations in progranulin cause tau-negative frontotemporal lobar degeneration with ubiquitin-positive inclusions. A major protein component of these inclusions is TDP-43, which becomes hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments, which apparently translocate from nuclei to the cytoplasm. Most progranulin mutations are nonsense mutations resulting in nonsensemediated mRNA decay and consequently reduced progranulin protein levels. However, some missense mutations are described that occur within the signal sequence and mature progranulin. We now demonstrate that a progranulin mutation located within the signal sequence (PGRN A9D) results in cytoplasmic missorting with extremely low expression. In contrast, two other progranulin mutations (PGRN P248L and R432C) are expressed as immature proteins but are inefficiently transported through and partially degraded within the secretory pathway, resulting in a significantly reduced secretion. Thus apparently all progranulin mutations cause reduced protein expression or secretion, although by different cellular mechanisms. To investigate a putative relationship between reduced expression of progranulin and TDP-43 relocalization and deposition, we down-regulated progranulin in human cell lines and in zebrafish. Upon reduction of progranulin, neither a major redistribution of TDP-43 nor proteolytic processing to disease-characterizing C-terminal fragments could be observed.Dementias are a major health problem in our aging society. The most frequent forms of dementia, namely Alzheimer disease, frontotemporal lobar degeneration (FTLD), 3 as well as dementia with Lewy bodies and related disorders are associated with selective neuronal cell loss. In these neurodegenerative disorders, proteins, which are normally soluble are known to misfold because of proteolytic processing and/or abnormal posttranslational modifications. Such insoluble amyloidogenic proteins are often deposited and may form reservoirs for neurotoxic oligomers (1). FTLD, which accounts approximately for 15% of all dementias, is characterized by two different types of cellular inclusions. About 40% of FTLD cases have tau-positive inclusions (2, 3). Genetic linkage led to the identification of more than 40 different mutations in the microtubule-associated protein tau gene locus on chromosome 17 (4). However, a number of familial FTLD cases failed to exhibit mutations in the tau gene, although strong linkage to chromosome 17 was observed (5). These cases were characterized by tau-and ␣-synuclein-negative, ubiquitin-positive cytoplasmic and intranuclear inclusions (3). The inclusions are observed in the frontotemporal cortex, the temporal neocortex, and the hippocampus and define the frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U). FTLD-U is the most frequent neuropathological form of FTLD and presents with progressive social, behavioral symptoms, and language dysfunction. Patients may also develop typical symptoms of motoneuron...

Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 + inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS-and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Doublehomozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism.neurodegeneration | zinc finger nuclease | proteomics A myotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin and TDP-43 + inclusions (FTLD-TDP) are incurable fatal neurodegenerative diseases. ALS is characterized by the loss of upper and lower motor neurons and FTLD patients suffer from degeneration of the frontal and temporal lobes. Both diseases belong to an ALS-FTLD disease spectrum (1, 2). TDP-43 is a RNA binding protein, which modulates RNA splicing, and systematic cross-linking and immunoprecipitation studies identified numerous RNA targets (3-5). About 90% of ALS cases are sporadic and the majority is pathologically characterized by insoluble TDP-43 inclusions (1, 6). Mutations in TDP-43 can lead to familial forms of ALS and FTLD-TDP (7,8). Most of the patientassociated mutations cluster in the C-terminal glycine-rich domain of TDP-43 (9), which mediates protein-protein interactions and is required for splicing-associated activities as well as autoregulation (10)(11)(12)(13)(14). Pathologically, ALS and FTLD-TDP are characterized by nuclear clearance and deposition of insoluble . Whether neurotoxicity of the TDP-43 inclusions or reduced TDP-43 function upon nuclear clearance is responsible for ALS and FTLD is under debate. However, little is known about the physiological function of TDP-43. To obtain insights into the in vivo function of TDP-43, we investigated the morphological, developmental, and molecular consequences of a loss of TDP-43 in zebrafish.

Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.

Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid b-peptide (Ab). Two proteases, b-and c-secretase proteolytically generate Ab from its precursor, the ß-amyloid precursor protein (APP). Inhibition of b-secretase, also referred to as beta-site APP cleaving enzyme (BACE1) or c-secretase is therefore of prime interest for the development of amyloid-lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nucleasemediated genome editing. bace1 mutants (bace1À/À) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1À/À. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1À/À. Double homozygous bace1À/À; bace2À/À fish do not enhance the single mutant phenotypes indicating non-redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 À/À associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo.

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BackgroundThe most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo.MethodWe generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence.ResultsZebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP.ConclusionThese novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0146-8) contains supplementary material, which is available to authorized users.