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.

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.

Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

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.

Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.

Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation.

Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.

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.

Spatial and temporal expression of RP58, a novel zinc finger transcriptional repressor, in mouse brain.

RP58, a novel zinc finger protein containing a POZ domain, is a sequence-specific transcriptional repressor. To understand the role of this protein, we examined RP58 gene expression in the developing mouse brain by quantitative polymerase chain reaction (PCR) and in situ hybridization. RP58 mRNA expression was detected at embryonic day (E) 10 in the neuroepithelium, and subsequently in the ventricular zones of the cerebral cortex in the E12 embryo. Strong expression was observed in the preplate in the cerebral cortex from this stage onward. High levels of expression continued to be detected in the cortical plate and subventricular zone of the neocortex, hippocampus, and parts of the amygdala, but not in the thalamus or striatum. These results suggest that RP58 plays a crucial role in neuronal proliferation, migration, and differentiation in the developing cerebral cortex. RP58 is also expressed in the adult mouse neocortex, hippocampus, parts of the amygdala, and granule cells in the cerebellum. Double in situ hybridization using GAD67 or VGLUT1 probes revealed that RP58 is expressed in glutamatergic excitatory neurons.

Two-photon excitation imaging of pancreatic islets with various fluorescent probes.

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.

Transfer of NMDAR2 cDNAs increases endogenous NMDAR1 protein and induces expression of functional NMDA receptors in PC12 cells.

The pheochromocytoma cell line (PC12) has been used as a model system for the study of regulation of expression of NMDA receptors. PC12 cells express a substantial amount of NMDAR1 subunit (NR1) mRNA, whereas they express only a small amount of NR1 protein. The level of functional NMDA receptor expression is almost negligible. To test the possibility that NMDAR2 subunits (NR2) control expression of functional NMDA receptors as well as NR1 protein, we transferred NR2A-D cDNAs into PC12 cells using adenovirus vectors. Prominent NMDA receptor-mediated currents were recorded in PC12 cells to which NR2A or NR2B cDNA was delivered without NR1 cDNA. The amplitudes of these responses were similar to those in PC12 cells to which NR1 cDNA was delivered together with NR2A or NR2B cDNA. In cells to which either NR2C or NR2D cDNA alone was delivered, NMDA receptor-mediated currents were also detected, although to a much lesser extent. These results showed that NR2 proteins produced by gene transfer are co-assembled with the endogenous NR1 protein to form functional heteromeric receptors. The delivery of NR2A-D cDNAs also increased the amount of NR1 protein but not that of NR1 mRNA, suggesting that this protein increase is due to post-transcriptional mechanisms. The effects of NR2A-B gene transfer on expression of NR1 protein were much more efficient than those of NR2C-D gene transfer.

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.