Expression analyses of C-terminal-binding protein 1 (CtBP1) during mouse brain development.

INTRODUCTION: CtBP1 (C-terminal-binding protein 1) is a multi-functional protein with well-established roles as a transcriptional co-repressor in the nucleus and a regulator of membrane fission in the cytoplasm. Although CtBP1 gene abnormalities have been reported to cause neurodevelopmental disorders, the physiological role and expression profile of CtBP1 remains to be elucidated. METHODS: In this study, we used biochemical, immunohistochemical and immunofluorescence methods to analyze the expression of CtBP1 during mouse brain development. RESULTS: Western blotting analyses revealed that CtBP1 appeared to be expressed mainly in the central nervous system throughout the developmental process. In immunohistochemical analyses, region-specific nuclear as well as weak cytoplasmic distribution of CtBP1 was observed in telencephalon at embryonic day (E)15 and E17. It is of note that CtBP1 was barely detected in axons, but observed in the nucleus of oligodendrocytes in the white matter at E17. As to cerebellum at postnatal day 30, CtBP1 appeared to be expressed in the nucleus and cytoplasm of Purkinje cells, the nucleus of granule cells and cells in the molecular layer (ML), and the ML per se where granule cell axons and Purkinje cell dendrites are enriched. In addition, CtBP1 was detected in the cerebellar nuclei. CONCLUSION: The obtained results suggest involvement of CtBP1 in brain function.

A Case of Cerebral Hyperperfusion Syndrome during Treatment of Transient Cerebral Vasospasm Following Carotid Artery Stenting.

Objective: We report a case in which transient cerebral vasospasm after carotid artery stenting (CAS) was effectively treated using arterial and intravenous infusion of fasudil hydrochloride, but cerebral hyperperfusion syndrome (CHS) developed during subsequent treatment. Case Presentation: The patient was a 79-year-old man who underwent right CAS to treat symptomatic right carotid artery stenosis. After the procedure, the patient developed left paresis and unilateral spatial neglect. The following day, he developed diffuse cerebral vasospasm in the right middle cerebral artery that improved immediately upon arterial infusion of fasudil hydrochloride. Intravenous infusion of fasudil hydrochloride was then started, but CHS with epileptic seizures developed after 1 day of treatment. After 23 days of medical treatment, the condition of the patient improved to mild hemiparesis. Conclusion: The present case suggests that transient cerebral vasospasm after CAS may turn into CHS during treatment and that continuous monitoring for cerebral perfusion is important.

Expression Analyses of Rich2/Arhgap44, a Rho Family GTPase-Activating Protein, during Mouse Brain Development.

Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.

Expression Analyses of Polo-Like Kinase 4, a Gene Product Responsible for Autosomal Recessive Microcephaly and Seckel Syndrome, during Mouse Brain Development.

Polo-like kinase 4 (Plk4) is a ser/thr kinase, which plays a central role in centriole duplication during the cell cycle. PLK4 gene abnormalities are responsible for autosomal recessive chorioretinopathy-microcephaly syndrome and Seckel syndrome. In this study, we performed expression analyses of Plk4 by focusing on mouse brain development. Western blotting analyses revealed that Plk4 with a molecular mass of ∼100 kDa was broadly expressed in adult mouse tissues with specific subcellular distribution. As to the central nervous system, Plk4 was expressed throughout the developmental process with drastic increase after P15, suggesting an essential role of Plk4 in differentiated neurons. In immunohistochemical analyses with mouse brain at embryonic day 14, Plk4 was detected dominantly at the cell-cell contact sites of neuronal progenitors in the ventricular zone. Plk4 was then diffusely distributed in the cell body of cortical neurons at P7, while it was enriched in the neuropil as well as soma of excitatory neurons in the cerebral cortex and hippocampus and Purkinje cells in the cerebellum at P30. Notably, biochemical fractionation analysis found an enrichment of Plk4 in the postsynaptic density fraction. Then, immunofluorescent analyses showed partial co-localization of Plk4 with excitatory synaptic markers, PSD95 and synaptophysin, in differentiated primary cultured hippocampal neurons. These results suggest that Plk4 takes part in the regulation of synaptic function in differentiated neurons.

Ischemic Stroke with Multiple Cerebral Artery Stenosis in a Patient with an Anaplastic Astrocytoma during Bevacizumab Treatment: A Case Report.

It has been reported that bevacizumab, an agent administered as an adjuvant therapy for high-grade gliomas, causes thromboembolic complications. We report a cerebral infarction with newly developed cerebral artery stenosis occurring during treatment with bevacizumab for an anaplastic astrocytoma. A 48-year-old female underwent excision surgery for an anaplastic astrocytoma on the right temporal lobe and received radiation therapy and chemotherapy with temozolomide. Twenty months after the maintenance therapy, treatment with bevacizumab was introduced for tumor recurrence. After the 14th course of bevacizumab at 6 months, 27 months after radiation therapy, the patient began experiencing mild right hemiparesis. Magnetic resonance imaging revealed scattered cerebral infarcts on the left frontal lobe and diffuse cerebral artery stenosis of the bilateral internal carotid artery system both inside and outside the radiation-treated area. Antiplatelet medication was commenced, and there was no recurrence of ischemic stroke. The morphological transition of the cerebral arteries should be carefully monitored via magnetic resonance angiography during post-radiation treatment with bevacizumab.

Expression Analyses of Cep152, a Responsible Gene Product for Autosomal Recessive Primary Microcephaly, during Mouse Brain Development.

Centrosomal protein 152 (Cep152) regulates centriole duplication as a molecular scaffold during the cell cycle. Its gene abnormalities are responsible for autosomal recessive primary microcephaly 9 and Seckel syndrome. In this study, we prepared an antibody against mouse Cep152, anti-Cep152, and performed expression analyses focusing on mouse brain development. Western blotting analyses revealed that Cep152 with a molecular mass of ∼150 kDa was expressed strongly at embryonic day (E)13 and then gradually decreased during the brain development process. Instead, protein bands of ∼80 kDa and ∼60 kDa came to be recognized after postnatal day (P)15 and P30, respectively. In immunohistochemical analyses, Cep152 was enriched in the centrosome of neuronal progenitors in the ventricular zone at E14, whereas it was diffusely distributed mainly in the cytoplasm of cortical neurons at P18. In developing cerebellum at P7, Cep152 was localized at the centrosome in the external granular layer, where neurogenesis takes place. Notably, biochemical analysis revealed that Cep152 was also present in the postsynaptic density fraction. Subsequent immunofluorescent analyses showed co-localization of Cep152 with excitatory synaptic markers, PSD95 and synaptophysin, but not with an inhibitory synaptic marker gephyrin in differentiated primary cultured hippocampal neurons. The obtained results suggest that Cep152 takes part not only in neurogenesis during corticogenesis but also in the regulation of synaptic function in differentiated neurons.

Expression Analyses of Rac3, a Rho Family Small GTPase, during Mouse Brain Development.

Rac3 is a member of Rho family small GTPases which regulate cellular signaling and cytoskeletal dynamics. The RAC3 gene abnormalities have been shown to cause neurodevelopmental disorders with structural brain anomalies, including polymicrogyria/dysgyria, callosal abnormalities, brainstem anomalies, and cerebellar dysplasia. Although this evidence indicates that Rac3 is essential in brain development, not only its molecular mechanism but also the expression profile is yet to be elucidated. In this study, we carried out expression analyses of Rac3 with mouse brain tissues. In immunoblotting, Rac3 exhibited a tissue-dependent expression profile in the young adult mouse and was expressed in a developmental stage-dependent manner in brain. In primary cultured hippocampal neurons, while Rac3 was distributed mainly in the cytoplasm, it was visualized in axon and dendrites with partial localization at synapses, in consistent with the observation in biochemical fractionation analyses. In immunofluorescence analyses with brain slices, Rac3 was distributed strongly and moderately in the axon and cytoplasm, respectively, of cerebral cortex at postnatal day (P) 2 and P18. Similar distribution profile was also observed in hippocampus. Taken together, the results obtained strongly suggest that Rac3 plays an important physiological role in neuronal tissues during corticogenesis, and defects in the Rac3 function induce structural brain anomalies leading to pathogenesis of neurodevelopmental disorders.

Physiological significance of WDR45, a responsible gene for ß-propeller protein associated neurodegeneration (BPAN), in brain development.

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause ß-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay with intellectual disability, the neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in excitatory synapses by biochemical fractionation. Since WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by in utero electroporation in mice to recapitulate the pathophysiological conditions of BPAN. Knockdown of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

The synaptic scaffolding protein CNKSR2 interacts with CYTH2 to mediate hippocampal granule cell development.

CNKSR2 is a synaptic scaffolding molecule that is encoded by the CNKSR2 gene located on the X chromosome. Heterozygous mutations to CNKSR2 in humans are associated with intellectual disability and epileptic seizures, yet the cellular and molecular roles for CNKSR2 in nervous system development and disease remain poorly characterized. Here, we identify a molecular complex comprising CNKSR2 and the guanine nucleotide exchange factor (GEF) for ARF small GTPases, CYTH2, that is necessary for the proper development of granule neurons in the mouse hippocampus. Notably, we show that CYTH2 binding prevents proteasomal degradation of CNKSR2. Furthermore, to explore the functional significance of coexpression of CNKSR2 and CYTH2 in the soma of granule cells within the hippocampal dentate gyrus, we transduced mouse granule cell precursors in vivo with small hairpin RNAs (shRNAs) to silence CNKSR2 or CYTH2 expression. We found that such manipulations resulted in the abnormal localization of transduced cells at the boundary between the granule cell layer and the hilus. In both cases, CNKSR2-knockdown and CYTH2-knockdown cells exhibited characteristics of immature granule cells, consistent with their putative roles in neuron differentiation. Taken together, our results demonstrate that CNKSR2 and its molecular interaction partner CYTH2 are necessary for the proper development of dentate granule cells within the hippocampus through a mechanism that involves the stabilization of a complex comprising these proteins.

Expression analyses of PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, during mouse brain development.

Abnormalities of PLEKHG2 gene, encoding a Rho family-specific guanine nucleotide exchange factor, are involved in microcephaly with intellectual disability. However, not only the role of PLEKHG2 in the developmental process but also its expression profile is unknown. In this study, we prepared a specific antibody against PLEKHG2 and carried out expression analyses with mouse tissues. In western blotting, PLEKHG2 exhibited a tissue-dependent expression profile in adult mouse and was expressed in a developmental stage-dependent manner in brain. Then, in immunohistochemical analyses, while PLEKHG2 was observed in the cortical plate and ventricular zone surface of the cerebral cortex at embryonic day 14, it came to be distributed throughout the cerebral cortex in layer II/III and V during corticogenesis. PLEKHG2 was also detected mainly in the nucleus of neurons in the hippocampal CA regions and dentate gyrus at P7. Notably, the nuclear accumulation disappeared at P30 and PLEKHG2 came to be located at the axons and/or dendrites at this time point. Moreover, in vitro immunofluorescence revealed that PLEKHG2 was at least partially localized at both excitatory and inhibitory synapses in primary cultured hippocampal neurons. These results suggest roles of PLEKHG2 in the development of the central nervous tissue and synaptic function.

Biochemical and morphological characterization of SEPT1 in mouse brain.

Septins are a highly conserved family of GTPases which are identified in diverse organisms ranging from yeast to humans. In mammals, nervous tissues abundantly contain septins and associations of septins with neurological disorders such as Alzheimer's disease and Parkinson's disease have been reported. However, roles of septins in the brain development have not been fully understood. In this study, we produced a specific antibody against mouse SEPT1 and carried out biochemical and morphological characterization of SEPT1. When the expression profile of SEPT1 during mouse brain development was analyzed by western blotting, we found that SEPT1 expression began to increase after birth and the increase continued until postnatal day 22. Subcellular fractionation of mouse brain and subsequent western blot analysis revealed the distribution of SEPT1 in synaptic fractions. Immunofluorescent analyses showed the localization of SEPT1 at synapses in primary cultured mouse hippocampal neurons. We also found the distribution of SEPT1 at synapses in mouse brain by immunohistochemistry. These results suggest that SEPT1 participates in various synaptic events such as the signaling, the neurotransmitter release, and the synapse formation/maintenance.

Role of Per3, a circadian clock gene, in embryonic development of mouse cerebral cortex.

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

Possible involvement of a cell adhesion molecule, Migfilin, in brain development and pathogenesis of autism spectrum disorders.

Migfilin, encoded by FBLIM1 at the 1p36 locus, is a multi-domain adaptor protein essential for various cellular processes such as cell morphology and migration. Small deletions and duplications at the 1p36 locus, monosomy of which results in neurodevelopmental disorders and multiple congenital anomalies, have also been identified in patients with autism spectrum disorder (ASD). However, the impact of FBLIM1, the gene within 1p36, on the pathogenesis of ASD is unknown. In this study, we performed morphological analyses of migfilin to elucidate its role in brain development. Migfilin was detected specifically in the embryonic and perinatal stages of the mouse brain. Either silencing or overexpression of migfilin in embryos following in utero electroporation disrupted Neocortical neuronal migration. Additionally, neurite elongation was impaired when migfilin was silenced in cultured mouse hippocampal neurons. We then screened FBLIM1 for rare exonic deletions/duplications in 549 Japanese ASD patients and 824 controls, detecting one case of ASD and intellectual delay that harbored a 26-kb deletion at 1p36.21 that solely included the C-terminal exon of FBLIM1. The FBLIM1 mRNA expression level in this case was reduced compared to levels in individuals without FBLIM1 deletion. Our findings indicate that tightly regulated expression of migfilin is essential for neuronal development and that FBLIM1 disruption may be related to the phenotypes associated with ASD and related neurodevelopmental disorders.

Reelin transiently promotes N-cadherin-dependent neuronal adhesion during mouse cortical development.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized ß-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

Role of Class III phosphoinositide 3-kinase in the brain development: possible involvement in specific learning disorders.

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD).

Secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity.

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

Reelin has a preventive effect on phencyclidine-induced cognitive and sensory-motor gating deficits.

Reelin has recently attracted attention because of its connection to several neuropsychiatric diseases. We previously reported the finding that prior transplantation of GABAergic neuron precursor cells into the medial prefrontal cortex (mPFC) of mice significantly prevented the induction of cognitive and sensory-motor gating deficits induced by phencyclidine (PCP). The majority of the precursor cells transplanted into the mPFC of the recipient mice differentiated into members of a somatostatin/Reelin-expressing class of GABAergic interneurons. These findings raised the possibility that Reelin secreted by the transplanted cells plays an important role in preventing the deficits induced by PCP. In this study, we investigated whether Reelin itself has a preventive effect on PCP-induced behavioral phenotypes by injecting conditioned medium containing Reelin into the lateral ventricle of the brains of 6- to 7-week-old male mice before administrating PCP. Behavioral analyses showed that the prior Reelin injection had a preventive effect against induction of the cognitive and sensory-motor gating deficits associated with PCP. Moreover, one of the types of Reelin receptor was found to be expressed by neurons in the mPFC. The results of this study point to the Reelin signaling pathway as a candidate target for the pharmacologic treatment of neuropsychiatric diseases.

Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS.

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (c9FTD/ALS). Recently, it was reported that an unconventional mechanism of repeat-associated non-ATG (RAN) translation arises from C9orf72 expansion. Sense and anti-sense transcripts of the expanded C9orf72 repeat, i.e. the dipeptide repeat protein (DRP) of glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA), are deposited in the brains of patients with c9FTD/ALS. However, the pathological significance of RAN-translated peptides remains unknown. We generated synthetic cDNAs encoding 100 repeats of DRP without a GGGGCC repeat and evaluated the effects of these proteins on cultured cells and cortical neurons in vivo. Our results revealed that the poly-GA protein formed highly aggregated ubiquitin/p62-positive inclusion bodies in neuronal cells. In contrast, the highly basic proteins poly-GR and PR also formed unique ubiquitin/p62-negative cytoplasmic inclusions, which co-localized with the components of RNA granules. The evaluation of cytotoxicity revealed that overexpressed poly-GA, poly-GP and poly-GR increased the substrates of the ubiquitin-proteasome system (UPS), including TDP-43, and enhanced the sensitivity to a proteasome inhibitor, indicating that these DRPs are cytotoxic, possibly via UPS dysfunction. The present data indicate that a gain-of-function mechanism of toxic DRPs possibly contributes to pathogenesis in c9FTD/ALS and that DRPs may serve as novel therapeutic targets in c9FTD/ALS.