No abstract

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.

Neurofibrillary tangles are one of the major pathological hallmarks of Alzheimer disease (AD).1 Neurofibrillary tangles are bundles of paired helical filament composed of the microtubule (MT)-associated protein tau in a hyperphosphorylated state (1, 2). Intracellular inclusions made of tau are also found in several other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), collectively called tauopathies (3, 4). Exonic and intronic mutations in the tau gene have recently been identified in familial FTDP-17 (5-7), indicating that dysfunction of the tau protein can cause the above mentioned neurodegenerative diseases. Many of the exonic mutations reduce the ability of tau to promote MT assembly (8 -10), but why these mutations cause the formation of tau inclusions is unknown.The tau protein in inclusions is hyperphosphorylated. Around 25 phosphorylation sites have been identified in paired helical filament tau from AD brains (11-13). Characteristic phosphorylation sites are Ser or Thr residues followed by Pro that are phosphorylated by the proline-directed protein kinase activities of extracellular signal-regulated kinase, glycogen synthase kinase 3␤ (GSK3␤), and cyclin-dependent kinase 5 (Cdk5). Phosphorylation reduces the ability of tau to bind to and polymerize MTs, resulting in an increase in the soluble form of tau dissociated from MTs. However, it is unclear how phosphorylated soluble tau assembles into filamentous aggregates of neurofibrillary tangles and then induces neurodegeneration. Elucidating the relationship between tau phosphorylation and aggregate formation is critical to our understanding of the pathogenesis. Phosphorylation of FTDP-17 mutant tau has been studied mainly with GSK3␤ or in non-neuronal cultured cells (14 -17). Mutant tau proteins were not phosphorylated more than wild-type (WT) tau either in transfected cultured cells or in vitro. Among them, R406W mutant tau showed significantly reduced phosphorylation in those experiments. However, both mutant tau and wildtype tau deposited in FTDP-17 brains are also hyperphosphorylated (18,19). It is important to resolve the discrepancy between the reduced phosphorylation of mutant tau in vitro and in cultured neurons and the high phosphorylation of mutant tau in pathological brains.Cdk5 is a proline-directed Ser/Thr kinase activated by a p35 or p39 Cdk5 activator (20 -22). Cdk5 activity is primarily detected in differentiated neurons because p35 and p39 show limited expression in neurons. As described above, Cdk5 is one of the tau protein kinases that phosphorylate tau in living neurons and is also able to generate several paired helical filament epitopes of tau in AD (23,24). However, the phosphorylation of FTDP-17 mutant tau by Cdk5 has not yet been examined. This question should be addressed because the involvement of Cdk5 in the pathogenic phosphorylation of tau has recently become more evident (25,26). In ...

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13- cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.

Jacobsen syndrome (JBS) is a rare congenital disorder caused by a terminal deletion of the long arm of chromosome 11. A subset of patients exhibit social behavioural problems that meet the diagnostic criteria for autism spectrum disorder (ASD); however, the underlying molecular pathogenesis remains poorly understood. PX-RICS is located in the chromosomal region commonly deleted in JBS patients with autistic-like behaviour. Here we report that PX-RICS-deficient mice exhibit ASD-like social behaviours and ASD-related comorbidities. PX-RICS-deficient neurons show reduced surface γ-aminobutyric acid type A receptor (GABAAR) levels and impaired GABAAR-mediated synaptic transmission. PX-RICS, GABARAP and 14-3-3ζ/θ form an adaptor complex that interconnects GABAAR and dynein/dynactin, thereby facilitating GABAAR surface expression. ASD-like behavioural abnormalities in PX-RICS-deficient mice are ameliorated by enhancing inhibitory synaptic transmission with a GABAAR agonist. Our findings demonstrate a critical role of PX-RICS in cognition and suggest a causal link between PX-RICS deletion and ASD-like behaviour in JBS patients.

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In our previous study, we identified RICS, a novel β β β β -catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.

We have recently shown that ␤-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a ␤-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-␤-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3 or -resulted in the disappearance of N-cadherin and ␤-catenin from the cellcell boundaries. Furthermore, we found that PX-RICS and 14-3-3/ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and ␤-catenin. Both RNAi-and dynamitin-mediated inhibition of dyneindynactin function also led to the absence of N-cadherin and ␤-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3/ complex links the N-cadherin-␤-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.In general, cargo proteins to be exported from the endoplasmic reticulum (ER) 2 have characteristic amino acid sequences called "ER export motifs" that facilitate their ER exit (1-4). Classic cadherins (simply referred to cadherins hereafter) (5-9), however, are known to have no functional ER export motifs, and their efficient ER exit requires complex formation with ␤-catenin at the ER immediately after cadherin synthesis (10 -12). We have recently shown that this ␤-catenin-facilitated ER export of cadherins requires PX-RICS, a ␤-catenininteracting GTPase-activating protein (GAP) for Cdc42 (13).PX-RICS is an alternatively spliced isoform of RICS (RhoGAP involved in the ␤-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling) and contains phox homology (PX) and Src homology 3 domains in its N-terminal region (14,15). RICS is expressed predominantly in neurons of the brain and localized to the growth cone and postsynaptic density, where it regulates neurite extension and presumably N-methyl-D-aspartate signaling (14). In contrast, PX-RICS is expressed in a wide variety of tissues and cell lines and localized to the ER and cis-Golgi (13, 15). Our recent study has revealed that PX-RICS facilitates ER-to-Golgi transport of the N-cadherin-␤-catenin complex through its direct interaction with ␤-catenin, Cdc42, ␥-aminobutyric acid type A receptor-associated protein (GABARAP) and phosphatidylinositol 4-phosphate (PI4P) (13). This finding suggests that PX-RICS is a key molecule in a novel intracellular transport system that is independent of known ER export motifs and provides a molecular basis that explains why the assembly of cadherins with ␤-catenin is essential for efficient ER exit of cadherins. However, the precise molecular mechanisms by which PX-RICS triggers ER exit of the N-cadherin-␤-catenin complex remain to be elucidated. To address this issue, we attempted to identify PX-RICS-interacting scaffold proteins involved in the PX-RICS-dependent forward transport mechanism. Here we report that PX-RICS and its novel binding partne...