Shinobu Hirai

RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex

Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.

The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1–4 genes in the developing cortex

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure, Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here, we report that a transcriptional factor, RP58, negatively regulates all four Id genes (Id1–Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild‐type cortical progenitors. Furthermore, Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally, we determined p57 as an effector gene of RP58‐Id‐mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self‐renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis.

Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43

Intracellular aggregates of phosphorylated TDP-43 are a major component of ubiquitin-positive inclusions in the brains of patients with frontotemporal lobar degeneration and ALS and are considered a pathological hallmark. Here, to gain insight into the mechanism of intracellular TDP-43 accumulation, we examined the relationship between phosphorylation and aggregation of TDP-43. We found that expression of a hyperactive form of casein kinase 1 δ (CK1δ1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 (ubiquitin- and p62-positive) in cultured neuroblastoma SH-SY5Y cells. Insoluble phosphorylated TDP-43 prepared from cells co-expressing TDP-43 and CK1δ1-317 functioned as seeds for TDP-43 aggregation in cultured cells, indicating that CK1δ1-317-induced aggregated TDP-43 has prion-like properties. A striking toxicity and alterations of TDP-43 were also observed in yeast expressing TDP-43 and CK1δ1-317. Therefore, abnormal activation of CK1δ causes phosphorylation of TDP-43, leading to the formation of cytoplasmic TDP-43 aggregates, which, in turn, may trigger neurodegeneration.

Distinct pathways leading to TDP-43-induced cellular dysfunctions

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component protein of inclusions found in brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the molecular mechanisms by which TDP-43 causes neuronal dysfunction and death remain unknown. Here, we report distinct cytotoxic effects of full-length TDP-43 (FL-TDP) and its C-terminal fragment (CTF) in SH-SY5Y cells. When FL-TDP was overexpressed in the cells using a lentiviral system, exogenous TDP-43, like endogenous TDP-43, was expressed mainly in nuclei of cells without any intracellular inclusions. However, these cells showed striking cell death, caspase activation and growth arrest at G2/M phase, indicating that even simple overexpression of TDP-43 induces cellular dysfunctions leading to apoptosis. On the other hand, cells expressing TDP-43 CTF showed cytoplasmic aggregates but without significant cell death, compared with cells expressing FL-TDP. Confocal microscopic analyses revealed that RNA polymerase II (RNA pol II) and several transcription factors, such as specificity protein 1 and cAMP-response-element-binding protein, were co-localized with the aggregates of TDP-43 CTF, suggesting that sequestration of these factors into TDP-43 aggregates caused transcriptional dysregulation. Indeed, accumulation of RNA pol II at TDP-43 inclusions was detected in brains of patients with FTLD-TDP. Furthermore, apoptosis was not observed in affected neurons of FTLD-TDP brains containing phosphorylated and aggregated TDP-43 pathology. Our results suggest that different pathways of TDP-43-induced cellular dysfunction may contribute to the degeneration cascades involved in the onset of ALS and FTLD-TDP.

Co-localization of a novel transcriptional repressor simiRP58 with RP58

We have cloned a novel transcriptional repressor protein, termed simiRP58, which has high homology to RP58. Both simiRP58 and RP58 belong to the POZ domain and Kruppel Zn finger (POK) family of proteins. Using the luciferase assay system, we found that simiRP58 also has transcriptional repressor activity like RP58. Northern blotting and quantitative RT-PCR showed that simiRP58 was expressed in testes at the highest level. In situ hybridization of testes showed that simiRP58 is expressed by spermatocytes in only a portion of the seminiferous tubules. In contrast, expression of RP58 by spermatocytes was ubiquitous in all seminiferous tubules. Using COS-7 cells, we observed that simiRP58 was localized in the cytoplasm, which is in contrast to RP58 that was localized in the nucleus. Interestingly, co-transfection with simiRP58 and RP58 induced changes in the localization patterns of both proteins.

Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.

AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate

Significance In mammals, AMPA-type glutamate receptors (GluAs) are expressed ubiquitously in the central nervous system and play critical roles in synaptic plasticity, learning, and memory. Here we examined GluAs in the ascidian, Ciona intestinalis, and determined that they are expressed in a limited subset of cells during early development. We further find that GluAs are required for development of the ocellus, a photoreceptive organ used during the swimming stage, and for tail resorption and body axis rotation during metamorphosis. These functions require ion influx through GluAs. This is a demonstration of an in vivo requirement for GluAs in organ formation and morphogenesis. GluAs are also expressed during mammalian development, suggesting that developmental roles of GluAs may be functionally conserved. AMPA-type glutamate receptors (GluAs) mediate fast excitatory transmission in the vertebrate central nervous system (CNS), and their function has been extensively studied in the mature mammalian brain. However, GluA expression begins very early in developing embryos, suggesting that they may also have unidentified developmental roles. Here, we identify developmental roles for GluAs in the ascidian Ciona intestinalis. Mammals express Ca2+-permeable GluAs (Ca-P GluAs) and Ca2+-impermeable GluAs (Ca-I GluAs) by combining subunits derived from four genes. In contrast, ascidians have a single gluA gene. Taking advantage of the simple genomic GluA organization in ascidians, we knocked down (KD) GluAs in Ciona and observed severe impairments in formation of the ocellus, a photoreceptive organ used during the swimming stage, and in resorption of the tail and body axis rotation during metamorphosis to the adult stage. These defects could be rescued by injection of KD-resistant GluAs. GluA KD phenotypes could also be reproduced by expressing a GluA mutant that dominantly inhibits glutamate-evoked currents. These results suggest that, in addition to their role in synaptic communication in mature animals, GluAs also have critical developmental functions.

C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are neurodegenerative diseases characterized by accumulation of insoluble aggregates of phosphorylated 43-kDa TAR DNA-binding protein (TDP-43), and linked with abnormal expansion of a hexanucleotide repeat in an intron of chromosome 9 open reading frame 72 (C9ORF72). However, the relationship between C9ORF72 mutations and TDP-43 aggregation remains unknown. Non-ATG-dependent translation of C9ORF72 repeats produces dipeptide repeat (DPR) proteins, which form p62-positive aggregates in cerebral cortex and cerebellum of patients. Here, we show that the formation of poly-GA protein inclusions induced intracellular aggregation of endogenous and exogenous TDP-43 in cultured cells. Poly-GA aggregation preceded accumulation of phosphorylated TDP-43. These inclusions induced intracellular aggregation of phosphorylated TDP-43, but not tau or alpha-synuclein. Formation of phosphorylated TDP-43 aggregates depends on the number of poly-GA repeats. Detergent-insoluble fraction from cells co-expressing poly-GA and TDP-43 could function as seeds for further TDP-43 aggregation. These findings suggest a novel pathogenic mechanism that poly-GA protein aggregation directly promotes pathogenic changes of TDP-43 without formation of nuclear RNA foci containing GGGGCC repeat expansion or loss-of-function of the C9ORF72 protein.

Benzodiazepines induce sequelae in immature mice with inflammation-induced status epilepticus.

OBJECTIVE Since benzodiazepines (BZPs) became clinically available for the treatment of status epilepticus (SE) in children, the incidence of neurological sequelae has increased. However, the cause-effect relationship is poorly understood. In this paper, we examined the effect of BZPs on an inflammation-induced SE (iSE) animal model. METHOD Inflammation was induced by injecting poly(I:C) (pIC 10 mg/kg, postnatal day 12-14), seizure was induced by injecting pilocarpine hydrochloride (PILO 200 mg/kg, postnatal day 15) into C57BL/6J mice, and the pIC+PILO mice were used as the iSE model (miSE). The GABA-A receptor agonist midazolam (MDL 0.5 mg/kg) was used to inhibit seizures. Sequelae were evaluated by performing behavior and immunohistochemical analyses in the chronic phase. RESULT The exploratory activity of mice in the miSE plus MDL group increased significantly, indicating that hyperactivity was newly induced by MDL in miSE mice. The contextual fear memory of the miSE mice was also significantly increased and that of miSE treated with MDL returned to the normal level. The parvalbumin-positive GABA neurons were decreased in number by pIC+PILO which was rescued by MDL. Apoptosis marker ssDNA-positive cells were increased by pIC+PILO which could not be rescued by MDL. Therefore, we propose that BZP-dependent therapy for SE needs to be rethought from the perspective of using other treatment approaches.