Cancer-specific epigenome identifies oncogenic hijacking by nuclear factor I family proteins for medulloblastoma progression.

Normal cells coordinate proliferation and differentiation by precise tuning of gene expression based on the dynamic shifts of the epigenome throughout the developmental timeline. Although non-mutational epigenetic reprogramming is an emerging hallmark of cancer, the epigenomic shifts that occur during the transition from normal to malignant cells remain elusive. Here, we capture the epigenomic changes that occur during tumorigenesis in a prototypic embryonal brain tumor, medulloblastoma. By comparing the epigenomes of the different stages of transforming cells in mice, we identify nuclear factor I family of transcription factors, known to be cell fate determinants in development, as oncogenic regulators in the epigenomes of precancerous and cancerous cells. Furthermore, genetic and pharmacological inhibition of NFIB validated a crucial role of this transcription factor by disrupting the cancer epigenome in medulloblastoma. Thus, this study exemplifies how epigenomic changes contribute to tumorigenesis via non-mutational mechanisms involving developmental transcription factors.

Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Control of Synaptic Levels of Nicotinic Acetylcholine Receptor by the Sequestering Subunit Dα5 and Secreted Scaffold Protein Hig.

The presentation of nicotinic acetylcholine receptors (nAChRs) on synaptic membranes is crucial for generating cholinergic circuits, some of which are associated with memory function and neurodegenerative disorders. Although the physiology and structure of nAChR, a cation channel comprising five subunits, have been extensively studied, little is known about how the receptor levels in interneuronal synapses are determined and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices and intracellular proteins. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts in the brain. Only the loss of function of Dα5 among the 10 nAChR subunits suppressed hig mutant phenotypes in both male and female flies. Dα5 behaved as a lethal factor when Hig was defective; loss of Dα5 in hig mutants rescued lethality, upregulating Dα6 synaptic levels. By contrast, levels of Dα5, Dα6, and Dα7 subunits were all reduced in hig mutants. These three subunits have distinct properties for interaction with Hig or trafficking, as confirmed by chimeric subunit experiments. Notably, the chimeric Dα5 protein, which has the extracellular sequences that display no positive interaction with Hig, exhibited abnormal distribution and lethality even in the presence of Hig. We propose that the sequestering subunit Dα5 functions by reducing synaptic levels of nAChR through internalization, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.SIGNIFICANCE STATEMENT Because the cholinergic synapse is one of the major synapses that generate various brain functions, numerous studies have sought to reveal the physiology and structure of the nicotinic acetylcholine receptor (nAChR). However, little is known about how synaptic levels of nAChR are controlled and which nAChR subunits participate in the regulatory process in cooperation with synaptic cleft matrices. By a genetic screen of Drosophila, we identified mutations in the nAChR subunit Dα5 gene as suppressors that restored the mutant phenotypes of hig, which encodes a secretory matrix protein localized to cholinergic synaptic clefts. Our data indicate that Dα5 functions in reducing synaptic levels of nAChR, and this process is blocked by Hig, which tethers Dα5 to the synaptic cleft matrix.

DNA damage in embryonic neural stem cell determines FTLDs' fate via early-stage neuronal necrosis.

The early-stage pathologies of frontotemporal lobal degeneration (FTLD) remain largely unknown. In VCPT262A-KI mice carrying VCP gene mutation linked to FTLD, insufficient DNA damage repair in neural stem/progenitor cells (NSCs) activated DNA-PK and CDK1 that disabled MCM3 essential for the G1/S cell cycle transition. Abnormal neural exit produced neurons carrying over unrepaired DNA damage and induced early-stage transcriptional repression-induced atypical cell death (TRIAD) necrosis accompanied by the specific markers pSer46-MARCKS and YAP. In utero gene therapy expressing normal VCP or non-phosphorylated mutant MCM3 rescued DNA damage, neuronal necrosis, cognitive function, and TDP43 aggregation in adult neurons of VCPT262A-KI mice, whereas similar therapy in adulthood was less effective. The similar early-stage neuronal necrosis was detected in PGRNR504X-KI, CHMP2BQ165X-KI, and TDPN267S-KI mice, and blocked by embryonic treatment with AAV-non-phospho-MCM3. Moreover, YAP-dependent necrosis occurred in neurons of human FTLD patients, and consistently pSer46-MARCKS was increased in cerebrospinal fluid (CSF) and serum of these patients. Collectively, developmental stress followed by early-stage neuronal necrosis is a potential target for therapeutics and one of the earliest general biomarkers for FTLD.

Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1.

During development, neural progenitors are in proliferative and immature states; however, the molecular machinery that cooperatively controls both states remains elusive. Here, we report that cyclin D1 (CCND1) directly regulates both proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates S309 of ATOH1, which inhibits additional phosphorylation at S328 and consequently prevents S328 phosphorylation-dependent ATOH1 degradation. Additionally, PROX1 downregulates Ccnd1 expression by histone deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. Moreover, WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferative and immature states of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which are suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

Design and synthesis of gene-directed caged cyclic nucleotides exhibiting cell type selectivity.

We designed a new caging group that can be photoactivated only in the presence of a non-endogenous enzyme when exposed to 405 nm light. Because cells or tissues can be genetically tagged by an exogenously expressed enzyme, this novel method can serve as a strategy for adding targeting abilities to photocaged compounds.

Role of the Drosophila YATA protein in the proper subcellular localization of COPI revealed by in vivo analysis.

yata mutants of Drosophila melanogaster exhibit phenotypes including progressive brain shrinkage, developmental abnormalities and shortened lifespan, whereas in mammals, null mutations of the yata ortholog Scyl1 result in motor neuron degeneration. yata mutation also causes defects in the anterograde intracellular trafficking of a subset of proteins including APPL, which is the Drosophila ortholog of mammalian APP, a causative molecule in Alzheimer's disease. SCYL1 binds and regulates the function of coat protein complex I (COPI) in secretory vesicles. Here, we reveal a role for the Drosophila YATA protein in the proper localization of COPI. Immunohistochemical analyses performed using confocal microscopy and structured illumination microscopy showed that YATA colocalizes with COPI and GM130, a cis-Golgi marker. Analyses using transgenically expressed YATA with a modified N-terminal sequence revealed that the N-terminal portion of YATA is required for the proper subcellular localization of YATA. Analysis using transgenically expressed YATA proteins in which the C-terminal sequence was modified revealed a function for the C-terminal portion of YATA in the subcellular localization of COPI. Notably, when YATA was mislocalized, it also caused the mislocalization of COPI, indicating that YATA plays a role in directing COPI to the proper subcellular site. Moreover, when both YATA and COPI were mislocalized, the staining pattern of GM130 revealed Golgi with abnormal elongated shapes. Thus, our in vivo data indicate that YATA plays a role in the proper subcellular localization of COPI.

AUTS2 Regulation of Synapses for Proper Synaptic Inputs and Social Communication.

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants.

We previously identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-coloured compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the Amyloid precursor protein-like protein, which is an orthologue of Amyloid precursor protein, which is a causative molecule of Alzheimer's disease. In this study, we examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. We found that abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies, but were observed in very aged red-eyed wild-type flies. Thus, our data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies.

Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model.

APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. We previously showed that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. In this study, we examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, we observed that synaptic transmission accuracy was impaired in our model, and this phenotype was improved by yata knockdown. Thus, our data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP.

Characterization of the activity of ß-galactosidase from Escherichia coli and Drosophila melanogaster in fixed and non-fixed Drosophila tissues.

ß-Galactosidase encoded by the Escherichia coli lacZ gene, is widely used as a reporter molecule in molecular biology in a wide variety of animals. ß-Galactosidase retains its enzymatic activity in cells or tissues even after fixation and can degrade X-Gal, a frequently used colormetric substrate, producing a blue color. Therefore, it can be used for the activity staining of fixed tissues. However, the enzymatic activity of the ß-galactosidase that is ectopically expressed in the non-fixed tissues of animals has not been extensively studied. Here, we report the characterization of ß-galactosidase activity in Drosophila tissues with and without fixation in various experimental conditions comparing the activity of two evolutionarily orthologous ß-galactosidases derived from the E. coli lacZ and Drosophila melanogaster DmelGal genes. We performed quantitative analysis of the activity staining of larval imaginal discs and an in vitro assay using larval lysates. Our data showed that both E. coli and Drosophila ß-galactosidase can be used for cell-type-specific activity staining, but they have their own preferences in regard to conditions. E. coli ß-galactosidase showed a preference for neutral pH but not for acidic pH compared with Drosophila ß-galactosidase. Our data suggested that both E. coli and Drosophila ß-galactosidase show enzymatic activity in the physiological conditions of living animals when they are ectopically expressed in a desired specific spatial and temporal pattern. This may enable their future application to studies of chemical biology using model animals.

Systems biology analysis of Drosophila in vivo screen data elucidates core networks for DNA damage repair in SCA1.

DNA damage repair is implicated in neurodegenerative diseases; however, the relative contributions of various DNA repair systems to the pathology of these diseases have not been investigated systematically. In this study, we performed a systematic in vivo screen of all available Drosophila melanogaster homolog DNA repair genes, and we tested the effect of their overexpression on lifespan and developmental viability in Spinocerebellar Ataxia Type 1 (SCA1) Drosophila models expressing human mutant Ataxin-1 (Atxn1). We identified genes previously unknown to be involved in CAG-/polyQ-related pathogenesis that function in multiple DNA damage repair systems. Beyond the significance of each repair system, systems biology analyses unraveled the core networks connecting positive genes in the gene screen that could contribute to SCA1 pathology. In particular, RpA1, which had the largest effect on lifespan in the SCA1 fly model, was located at the hub position linked to such core repair systems, including homologous recombination (HR). We revealed that Atxn1 actually interacted with RpA1 and its essential partners BRCA1/2. Furthermore, mutant but not normal Atxn1 impaired the dynamics of RpA1 in the nucleus after DNA damage. Uptake of BrdU by Purkinje cells was observed in mutant Atxn1 knockin mice, suggesting their abnormal entry to the S-phase. In addition, chemical and genetic inhibitions of Chk1 elongated lifespan and recovered eye degeneration. Collectively, we elucidated core networks for DNA damage repair in SCA1 that might include the aberrant usage of HR.

Systematic analysis of fly models with multiple drivers reveals different effects of ataxin-1 and huntingtin in neuron subtype-specific expression.

The fruit fly, Drosophila melanogaster, is a commonly used model organism for neurodegenerative diseases. Its major advantages include a short lifespan and its susceptibility to manipulation using sophisticated genetic techniques. Here, we report the systematic comparison of fly models of two polyglutamine (polyQ) diseases. We induced expression of the normal and mutant forms of full-length Ataxin-1 and Huntingtin exon 1 in cholinergic, dopaminergic, and motor neurons, and glial cells using cell type-specific drivers. We systematically analyzed their effects based on multiple phenotypes: eclosion rate, lifespan, motor performance, and circadian rhythms of spontaneous activity. This systematic assay system enabled us to quantitatively evaluate and compare the functional disabilities of different genotypes. The results suggest different effects of Ataxin-1 and Huntingtin on specific types of neural cells during development and in adulthood. In addition, we confirmed the therapeutic effects of LiCl and butyrate using representative models. These results support the usefulness of this assay system for screening candidate chemical compounds that modify the pathologies of polyQ diseases.

A functional deficiency of TERA/VCP/p97 contributes to impaired DNA repair in multiple polyglutamine diseases.

It is hypothesized that a common underlying mechanism links multiple neurodegenerative disorders. Here we show that transitional endoplasmic reticulum ATPase (TERA)/valosin-containing protein (VCP)/p97 directly binds to multiple polyglutamine disease proteins (huntingtin, ataxin-1, ataxin-7 and androgen receptor) via polyglutamine sequence. Although normal and mutant polyglutamine proteins interact with TERA/VCP/p97, only mutant proteins affect dynamism of TERA/VCP/p97. Among multiple functions of TERA/VCP/p97, we reveal that functional defect of TERA/VCP/p97 in DNA double-stranded break repair is critical for the pathology of neurons in which TERA/VCP/p97 is located dominantly in the nucleus in vivo. Mutant polyglutamine proteins impair accumulation of TERA/VCP/p97 and interaction of related double-stranded break repair proteins, finally causing the increase of unrepaired double-stranded break. Consistently, the recovery of lifespan in polyglutamine disease fly models by TERA/VCP/p97 corresponds well to the improvement of double-stranded break in neurons. Taken together, our results provide a novel common pathomechanism in multiple polyglutamine diseases that is mediated by DNA repair function of TERA/VCP/p97.

A restricted level of PQBP1 is needed for the best longevity of Drosophila.

A number of neurological diseases are caused by mutations of RNA metabolism-related genes. A complicating issue is that whether under- or overfunction of such genes is responsible for the phenotype. Polyglutamine tract binding protein-1, a causative gene for X-linked mental retardation, is also involved in RNA metabolism, and both mutation and duplication of the gene were reported in human patients. In this study, we first report a novel phenotype of dPQBP1 (drosophila homolog of Polyglutamine tract binding protein-1)-mutant flies, lifespan shortening. We next address the gene dose-phenotype relationship in lifespan shortening and in learning disability, a previously described phenotype. The 2 phenotypes are rescued by dPQBP1 but in different dose-phenotype relationships. Either insufficient or excessive expression of dPQBP1 does not recover lifespan, while excessive expression recovers learning ability. We finally address the mechanism of lifespan shortening. Tissue-specific expression of dPQBP1-RNA interference construct reveals both neural and nonneural dPQBP1 contribute to the lifespan, while the latter has a dominant effect. Gene expression profiling suggested retinophilin/MORN repeat containing 4, a gene promoting axonal degeneration, to contribute to lifespan shortening by neural dPQBP1. Systems biology analysis of the gene expression profiles revealed indirect influence of dPQBP1 on insulin-like growth factor 1, insulin receptor, and peroxisome proliferator-activated receptorα/γ signaling pathways in nonneural tissues. Collectively, given that dPQBP1 affects multiple pathways in different dose-dependent and tissue-specific manners, dPQBP1 at a restricted expression level is needed for the best longevity.

Ku70 alleviates neurodegeneration in Drosophila models of Huntington's disease.

DNA damage accumulates in genome DNA during the long life of neurons, thus DNA damage repair is indispensable to keep normal functions of neurons. We previously reported that Ku70, a critical molecule for DNA double strand break (DSB) repair, is involved in the pathology of Huntington's disease (HD). Mutant huntingtin (Htt) impaired Ku70 function via direct interaction, and Ku70 supplementation recovered phenotypes of a mouse HD model. In this study, we generate multiple Drosophila HD models that express mutant huntingtin (Htt) in eye or motor neuron by different drivers and show various phenotypes. In such fly models, Ku70 co-expression recovers lifespan, locomotive activity and eye degeneration. In contrast, Ku70 reduction by heterozygous null mutation or siRNA-mediated knock down accelerates lifespan shortening and locomotion disability. These results collectively support that Ku70 is a critical mediator of the HD pathology and a candidate therapeutic target in HD.

Drosophila PQBP1 regulates learning acquisition at projection neurons in aversive olfactory conditioning.

Polyglutamine tract-binding protein-1 (PQBP1) is involved in the transcription-splicing coupling, and its mutations cause a group of human mental retardation syndromes. We generated a fly model in which the Drosophila homolog of PQBP1 (dPQBP1) is repressed by insertion of piggyBac. In classical odor conditioning, learning acquisition was significantly impaired in homozygous piggyBac-inserted flies, whereas the following memory retention was completely normal. Mushroom bodies (MBs) and antennal lobes were morphologically normal in dPQBP1-mutant flies. Projection neurons (PNs) were not reduced in number and their fiber connections were not changed, whereas gene expressions including NMDA receptor subunit 1 (NR1) were decreased in PNs. Targeted double-stranded RNA-mediated silencing of dPQBP1 in PNs, but not in MBs, similarly disrupted learning acquisition. NR1 overexpression in PNs rescued the learning disturbance of dPQBP1 mutants. HDAC (histone deacetylase) inhibitors, SAHA (suberoylanilide hydroxamic acid) and PBA (phenylbutyrate), that upregulated NR1 partially rescued the learning disturbance. Collectively, these findings identify dPQBP1 as a novel gene regulating learning acquisition at PNs.

Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity.

Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple alpha-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1.

Mutant huntingtin impairs Ku70-mediated DNA repair.

DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt-expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington's disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology.