Strumpellin/WASHC5 regulates the structural plasticity of cortical neurons involved in gait coordination.

Strumpellin/Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex subunit 5 (WASHC5) is a core component of the WASH complex, and its mutations confer pathogenicity for hereditary spastic paraplegia (HSP) type SPG8, a rare neurodegenerative gait disorder. WASH complex activates actin-related protein-2/3-mediated actin polymerization and plays a pivotal role in intracellular membrane trafficking in endosomes. In this study, we examined the role of strumpellin in the regulation of structural plasticity of cortical neurons involved in gait coordination. Administration of a lentivirus containing a strumpellin-targeting short hairpin RNA (shRNA) to cortical motor neurons lead to abnormal motor coordination in mice. Strumpellin knockdown using shRNA attenuated dendritic arborization and synapse formation in cultured cortical neurons, and this effect was rescued by wild-type strumpellin expression. Compared with the wild-type, strumpellin mutants N471D or V626F identified in patients with SPG8 exhibited no differences in rescuing the defects. Moreover, the number of F-actin clusters in neuronal dendrites was decreased by strumpellin knockdown and rescued by strumpellin expression. In conclusion, our results indicate that strumpellin regulates the structural plasticity of cortical neurons via actin polymerization.

Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis.

PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.

Phosphoproteome profiling of hippocampal synaptic plasticity.

The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and dephosphorylation of neuronal proteins are regulated by synaptic plasticity, no systematic analyses of this have yet been conducted. We performed a phosphoproteomic analysis of hippocampal synaptic plasticity using a nano-Acquity/Synapt LC-MS/MS system. Neuronal proteins were extracted from hippocampal tissues and cultured neurons exposed to long-term potentiation (LTP) or long-term depression (LTD). Filter-aided sample preparation (FASP) was performed to remove residual anionic detergents for complete tryptic digestion. Phosphopeptides were then enriched using TiO2 chromatography, followed by immunoaffinity chromatography with an anti-phosphotyrosine antibody. Among the 1500 phosphopeptides identified by LC-MS/MS, 374 phosphopeptides were detected simultaneously in both hippocampal tissues and cultured neurons. Semi-quantification counting the number of spectra of each phosphopeptide showed that 42 of 374 phosphopeptides changed significantly depending on synaptic plasticity. In conclusion, a new proteomic method using sequential enrichment of phosphopeptides and semi-quantification enabled the phosphoproteomic analysis of hippocampal synaptic plasticity.

CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons.

For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.

Nanoplasmonic immunosensor for the detection of SCG2, a candidate serum biomarker for the early diagnosis of neurodevelopmental disorder.

The neural circuits of the infant brain are rapidly established near 6 months of age, but neurodevelopmental disorders can be diagnosed only at the age of 2-3 years using existing diagnostic methods. Early diagnosis is very important to alleviate life-long disability in patients through appropriate early intervention, and it is imperative to develop new diagnostic methods for early detection of neurodevelopmental disorders. We examined the serum level of secretogranin II (SCG2) in pediatric patients to evaluate its potential role as a biomarker for neurodevelopmental disorders. A plasmonic immunosensor performing an enzyme-linked immunosorbent assay (ELISA) on a gold nanodot array was developed to detect SCG2 in small volumes of serum. This nanoplasmonic immunosensor combined with tyramide signal amplification was highly sensitive to detect SCG2 in only 5 µL serum samples. The analysis using the nanoplasmonic immunosensor revealed higher serum SCG2 levels in pediatric patients with developmental delay than in the control group. Overexpression or knockdown of SCG2 in hippocampal neurons significantly attenuated dendritic arborization and synaptic formation. These results suggest that dysregulated SCG2 expression impairs neural development. In conclusion, we developed a highly sensitive nanoplasmonic immunosensor to detect serum SCG2, a candidate biomarker for the early diagnosis of neurodevelopmental disorders.

Autosomal dominant transmission of complicated hereditary spastic paraplegia due to a dominant negative mutation of KIF1A, SPG30 gene.

KIF1A is a brain-specific anterograde motor protein that transports cargoes towards the plus-ends of microtubules. Many variants of the KIF1A gene have been associated with neurodegenerative diseases and developmental delay. Homozygous mutations of KIF1A have been identified in a recessive subtype of hereditary spastic paraplegia (HSP), SPG30. In addition, KIF1A mutations have been found in pure HSP with autosomal dominant inheritance. Here we report the first case of familial complicated HSP with a KIF1A mutation transmitted in autosomal dominant inheritance. A heterozygous p.T258M mutation in KIF1A was found in a Korean family through targeted exome sequencing. They displayed phenotypes of mild intellectual disability with language delay, epilepsy, optic nerve atrophy, thinning of corpus callosum, periventricular white matter lesion, and microcephaly. A structural modeling revealed that the p.T258M mutation disrupted the binding of KIF1A motor domain to microtubules and its movement along microtubules. Assays of peripheral accumulation and proximal distribution of KIF1A motor indicated that the KIF1A motor domain with p.T258M mutation has reduced motor activity and exerts a dominant negative effect on wild-type KIF1A. These results suggest that the p.T258M mutation suppresses KIF1A motor activity and induces complicated HSP accompanying intellectual disability transmitted in autosomal dominant inheritance.