Norihito Uemura

Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44–100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1Δ7/Δ7 fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator‐like effector nucleases in medaka

Genome editing using engineered nucleases such as transcription activator‐like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN‐induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high‐throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5′ T, upstream of each TALEN‐binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.

ATP13A2 deficiency induces a decrease in cathepsin D activity, fingerprint-like inclusion body formation, and selective degeneration of dopaminergic neurons

Here, we report that normal ATP13A2 localizes in the lysosome, whereas disease‐associated variants remain in the endoplasmic reticulum. Cathepsin D activity was decreased in ATP13A2‐knockdown cells that displayed lysosome‐like bodies characterized by fingerprint‐like structures. Furthermore, an atp13a2 mutation in medaka fish resulted in dopaminergic neuronal death, decreased cathepsin D activity, and fingerprint‐like structures in the brain. Based on these results, lysosome abnormality is very likely to be the primary cause of KRS/PARK9.

PINK1 and Parkin complementarily protect dopaminergic neurons in vertebrates

Parkinsons disease (PD) is a common neurodegenerative disorder characterized by selective dopaminergic cell loss in the substantia nigra, but its pathogenesis remains unclear. The recessively inherited familial PD genes PARK2 and PARK6 have been attributed to mutations in the Parkin and PTEN-induced kinase 1 (PINK1) genes, respectively. Recent reports suggest that PINK1 works upstream of Parkin in the same pathway to regulate mitochondrial dynamics and/or conduct autophagic clearance of damaged mitochondria. This phenomenon is preserved from Drosophila to human cell lines but has not been demonstrated in a vertebrate animal model in vivo. Here, we developed a medaka fish (Oryzias latipes) model that is deficient in Pink1 and Parkin. We found that despite the lack of a conspicuous phenotype in single mutants for Pink1 or Parkin, medaka that are deficient in both genes developed phenotypes similar to that of human PD: late-onset locomotor dysfunction, a decrease in dopamine levels and a selective degeneration of dopaminergic neurons. Further analysis also revealed defects in mitochondrial enzymatic activity as well as cell death. Consistently, PINK1 and Parkin double-deficient MEF showed a further decrease in mitochondrial membrane potential and mitochondrial complex I activity as well as apoptosis compared with single-deficient MEF. Interestingly, these mitochondrial abnormalities in Parkin-deficient MEF were compensated by exogenous PINK1, but not by disease-related mutants. These results suggest that PINK1 and Parkin work in a complementary way to protect dopaminergic neurons by maintaining mitochondrial function in vertebrates.

Viable Neuronopathic Gaucher Disease Model in Medaka (Oryzias latipes) Displays Axonal Accumulation of Alpha-Synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

The first Japanese case of leukodystrophy with ovarian failure arising from novel compound heterozygous AARS2 mutations.

Even now, only a portion of leukodystrophy patients are correctly diagnosed, though various causative genes have been identified. In the present report, we describe a case of adult-onset leukodystrophy in a woman with ovarian failure. By whole-exome sequencing, a compound heterozygous mutation consisting of NM_020745.3 (AARS2_v001):c.1145C>A and NM_020745.3 (AARS2_v001):c.2255+1G>A was identified. Neither of the mutations has been previously reported, and this is the first report of alanyl-transfer RNA synthetase 2 mutation in Asia. We anticipate that further studies of the molecular basis of leukodystrophy will provide insight into its pathogenesis and hopefully lead to sophisticated diagnostic and treatment strategies.

Alpha-synuclein fibrils propagate through tunneling nanotubes

Intracellular Lewy bodies (LBs) or neurites (LNs) of misfolded alpha-synuclein (a-Syn) are molecular hallmarks of synucleinopathies such as Parkinson’s disease and dementia with Lewy bodies. Several lines of evidence suggest that LB/LN pathology propagates through neural pathways over time; however, the molecular mechanisms of cell-to-cell transmission of pathological a-Syn remain unknown. Here, Abounit and colleagues reported that tunneling nanotube (TNT) is one of the vital pathways of a-Syn propagation. Intercellular communication is essential for maintaining the function of multicellular organs. TNT, originally described by Rustom and colleagues in 2004, is F-actin-based intercellular membranous bridge with diameter of 50 to 200 nanometers. TNTs can be observed in various cell types, including neurons, and through which various cargoes, such as organelles, membrane vesicles, and pathogens (bacteria, human immunodeficiency virus, and prion, etc.), and even Ca signals, are transferred. Moreover, recent reports demonstrated that huntingtin and transactive response DNA binding protein 43 kDa may also be transmitted through TNT. In this report, Abounit and colleagues first demonstrated that preformed a-Syn fibrils tagged with fluorescent protein were taken in by neuron-like CAD cells and primary neurons (donor cells), which were transmitted to cocultured healthy cells (acceptor cells). Then, they examined how a-Syn fibrils were transmitted from donor cells to healthy acceptor cells. Although coculture of donor cells and acceptor cells induced considerable transmission of a-Syn, fewer a-Syn fibrils were observed in acceptor cells cultured alone in conditioned medium from donor cells and in acceptor cells cultured in sparse cell condition, suggesting that direct contact with donor cells facilitated cell-to-cell transmission. Following these data, they showed the images of fluorescent aSyn fibrils in TNTs between donor cells and acceptor cells by confocal microscopy. Furthermore, they demonstrated that transferred a-Syn aggregates are involved in lysosomal vesicles, possibly indicating that lysosomes are too overloaded to decompose the fibrils within the cell. This article raised the possibility that cell-to-cell transmission through TNT is one of the common mechanisms of prion-like propagation of misfolded amyloid proteins. To verify the involvement of TNT in the progression of neurodegenerative diseases, in vivo demonstration of transmission of pathological proteins through TNTs should be considered. However, it must be challenging because TNTs are fragile and there are no specific TNT markers. The significance of this study should be emphasized, but further research is required to clarify the mechanism of cell-to-cell transmission of pathological proteins.

Exploring the Pathogenetic Mechanisms underlying Parkinson's Disease in Medaka Fish

Teleost fish have recently been employed as a model for human neurodegenerative diseases. We used toxin exposure and genetic engineering to develop models of Parkinsons disease (PD) in the teleost fish, medaka. Among the toxins examined, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors, and tunicamycin all induced important features of PD in medaka. Specifically, these agents induced dopaminergic cell loss and reduced spontaneous movement, and the latter three toxins produced inclusion bodies that were ubiquitously distributed in the medaka brain. Despite the extensive distribution of these inclusion bodies, the middle diencephalic dopaminergic neurons were particularly susceptible to the effect of the toxins, suggesting that this cluster of dopaminergic neurons is analogous to the human substantia nigra. We have also created a variety of different genetic models using the Targeting Induced Local Lesions in Genomes (TILLING) method, and found that neither PTEN-induced putative kinase 1 (PINK1) mutants nor Parkin mutants disclosed significant dopaminergic cell loss. Surprisingly however, PINK1 and Parkin double mutants exhibited selective dopamine cell loss, as well as aggregation and deficit of mitochondrial activity. Another mutant, the ATP13A2 mutant, also expressed a PD phenotype, exhibiting marked cathepsin D reduction and fingerprint-like structures that are generally found in lysosome storage diseases. Taken together, these data indicate that medaka fish can serve as a new animal model for PD. In this review, we summarize our data and discuss the potential for future application of this animal model.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

Parkinsons disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinsons disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinsons disease.

Innate immunity regulates α-synuclein clearance

Intraneuronal deposition of a-synuclein, called a Lewy body, is the molecular hallmark of Parkinson’s disease (PD)/ dementia with Lewy bodies (DLB). The mechanisms of asynuclein aggregation are still unclear, but autophagy, a lysosome-dependent cellular degradation system, has an important role in clearance of a-synuclein in neurons. Lee et al previously showed that disturbance of the autophagylysosome system resulted in the accumulation of a-synuclein aggregates. Despite the importance of autophagy in protein homeostasis, little is known about the extracellular factors that regulate accumulation of a-synuclein and how autophagy is modulated in neurons. Delgado et al showed that Toll-like receptor (TLR) activation induced autophagy in immune cells. TLRs, pattern recognition receptors, are key players in the innate immunity. Autophagy in immune cells may result in direct elimination of microbes or assist cytosolic antigen delivery to major histocompatibility complex II processing. Kim et al have already reported that TLR2 was upregulated in PD and DLB patients as well as in PD model animals. In addition, they showed that oligometric a-synuclein induced neuroinflammation through activation of TLR2 in microglia. Several previous studies investigated the relationship between neurodegenerative diseases and TLRs; however, they were mainly focused on neuroinflammation caused by activated microglia. In this study, Kim et al generated A53T1Tlr2-/mice by crossing transgenic mice expressing human A53T asynuclein (A53T1) and Tlr2-knockout mice (Tlr2-/-) to gain a comprehensive understanding of the pathological roles of TLR2 in PD. Accumulation of a-synuclein was significantly reduced in neurons of A53T1Tlr2-/mice compared with A53T1 mice. In addition, knockdown of TLR2 by lentiviral shRNA ameliorated a-synuclein pathology and motor symptoms in A53T1 mice. Moreover, TLR2 activation inhibited autophagy and resulted in the accumulation of a-synuclein aggregates in in vitro neuronal models. Impairment of autophagy by TLR2 activation occurred through the AKT/ mTOR pathway. Because they previously reported a-synuclein oligomers as an agonist of TLR2 in microglia, these findings allow speculation that extracellular a-synuclein oligomers may inhibit autophagy through TLR2 in neurons and result in promoting accumulation of a-synuclein, which forms a vicious cycle. This study showed that TLR2 regulated autophagy in neurons, which could be a new therapeutic target for autophagy activation strategies. It is interesting that TLR2 activation induces autophagy to clear the pathogens in immune cells, whereas TLR2 activation in neurons has a completely different effect. Although it was reported that neurons express TLRs other than TLR2, their physiological functions are still largely unknown. Further research is needed to determine the pathological role of TLRs in neurodegenerative diseases including PD/DLB.