Abnormal Local Translation in Dendrites Impairs Cognitive Functions in Neuropsychiatric Disorders
Neurodegenerative disorders such as Huntington’ disease and frontotemporal lobar degeneration (FTLD) are often caused by aggregation of causative proteins and co-morbid with mental disorders of unknown origin. Although toxic protein aggregates may entrap proteins involved in psychiatric disorders and elicit deleterious effects, candidate mechanisms for this co-aggregation hypothesis remain unknown. Here we show a novel cytosolic protein aggregate in FTLD brain composed of DISC1, a biological mediator for mental illnesses and TDP-43, the major protein constituent of insoluble inclusions in FTLD. FTLD model mice with DISC1/TDP-43 co-aggregates in frontal cortex showed hyperactivity and social interaction deficits that were rescued by exogenous DISC1 expression. At the cellular level, the amounts of newly synthesized proteins in dendrites are reduced in the neurons that lack DISC1 expression or contain DISC1/TDP-43 co-aggregates. These results support our novel hypothesis that co-aggregation of risk factors for psychiatric diseases may compromise local translation in dendrites and mental conditions.
KeywordsProtein aggregation Protein misfolding TDP-43 FTLD Neuropsychiatric disease
Neurodegenerative disorders are associated with a wide spectrum of behavioral abnormalities including sensory, motor, memory and mental deficits (Orr and Zoghbi 2007). The neurodegenerative disease, frontotemporal lobar degeneration (FTLD), is characterized by neuronal atrophy in frontal and temporal brain lobes associated with marked deficits in cognitive behavior (Rascovsky et al. 2011; Irish et al. 2012). In FTLD brain, TAR DNA-binding protein 43 (TDP-43) is a major protein component of ubiquitin-positive hyper-phosphorylated inclusions (Neumann et al. 2006), suggesting that its aggregation is implicated in FTLD pathogenesis (Cohen et al. 2011; Ling et al. 2013). TDP-43 is a heterogeneous ribonucleoprotein containing two highly conserved RNA recognition motifs that are believed to selectively regulate RNA processing, such as exon splicing, mRNA transport and microRNA biogenesis (Lee et al. 2012) In neurons, TDP-43 is a constituent of neuronal RNA granules in dendrites (Lee et al. 2012; Wang et al. 2008) where it interacts with a complex of RNA binding proteins including fragile X mental-retardation protein (FMRP) and Staufen1, which collectively regulate mRNA transport and local translation (Wang et al. 2008), the processes that are critical for synaptic function and plasticity (Bramham and Wells 2007; Holt and Schuman 2013; Buffington et al. 2014). Considerable progress has been made to understand the molecular mechanism of neurodegeneration caused by TDP-43 aggregation (Baralle et al. 2013). However, it remains unclear how TDP-43 aggregation mediates complex behavioral phenotypes in FTLD including progressive impairment in mental function (Rascovsky et al. 2011; Irish et al. 2012). In this study, we investigate the causal mechanism for psychiatric symptoms in FTLD by testing the general hypothesis that, at least in some neurodegenerative diseases, co-aggregation of casual disease factors like TDP-43, with genetic risk factors for psychiatric disease may alter cellular and behavioral phenotypes, resulting in overt manifestation of psychiatric symptoms.
18.2.1 TDP-43 Forms Co-Aggregates with DISC1 in Neurons
Several RNA-binding proteins such as TDP-43, FUS/TLS and FMRP were co-immunoprecipitated with DISC1. So, we asked whether RNA is critical for this interaction. To examine this possibility, we treated mouse brain homogenates with or without RNase, followed by co-immunoprecipitation with an anti-DISC1 antibody. RNase treatment abolished the binding of DISC1 to RNA-binding proteins, indicating RNA-dependence of the DISC1 association. This result suggests that DISC1 has a functional role in RNA metabolism to regulate neuronal functions. Furthermore, we investigated whether DISC1 regulates the abundance of synaptic proteins. When we examined amounts of postsynaptic proteins in synaptosomal fractions by western blotting, we found that the DISC1 depletion reduced levels of several key postsynaptic proteins in dendrites.
18.2.2 Role of DISC1 in Local Translation in Dendrites
To investigate whether the reduced levels of synaptic proteins in synaptosomal fractions might be caused by impaired local translation in dendrites, we examined the involvement of DISC1 in mRNA translation. Previous studies indicated that TDP-43 is associated with a translational machinery and polyribosomes (Higashi et al. 2013; Coyne and Siddegowda 2014). DISC1 can interact with TDP-43, therefore we examined whether DISC1 is also associated with a translational machinery by polysome gradient centrifugation analysis. DISC1 was detected in polyribosome fractions, as observed for TDP-43 (Coyne and Siddegowda 2014). After either EDTA or RNase treatment (Stefani et al. 2004), DISC1 in the polyribosome fractions co-migrated with TDP-43 and ribosomal S6 (RS6), indicating that, like TDP-43, DISC1 interacts with polyribosomes. Immunoprecipitation experiments demonstrated that DISC1 forms a complex with TDP-43 in the polyribosome fraction and that this binding was more significant in cerebral cortex than that cerebellum. Together, these data suggest that the DISC1-TDP-43 interaction is crucial for translation in cerebral cortex.
To further investigate the possible roles of DISC1 in local translation in dendrites, we examined whether DISC1 regulates translation of synaptic mRNAs in neurons by Surface Sensing of Translation (SUnSET), a method to monitor new protein synthesis by incorporation of puromycin into newly synthesized polypeptides (Schmidt et al. 2009). First, we assessed whether DISC1 might regulate local translation of synaptic mRNAs which are bound with DISC1. We labeled newly synthesized proteins in cultured cortical neurons with puromycin, and puromycin-incorporated proteins were immunoprecipitated from the synaptosomal fraction, followed by detection of newly synthesized postsynaptic NR2B and PSD95 proteins by western blotting. Remarkably, DISC1 knockdown significantly reduced synthesis of these synaptic proteins, which were restored by co-expression of an RNAi-resistant form of DISC1. These results suggest that DISC1 plays a critical role in dendrite local translation.
18.2.3 TDP-43-DISC1 Co-Aggregation Inhibits Local Translation in Dendrites
Since endogenous DISC1 is readily sequestered into TDP-220C aggregates, we hypothesized that the co-aggregation between DISC1 and TDP-220C might impair local translation, similar to the DISC1 depletion. Overexpression of TDP-220C in neurons decreased synaptic protein levels in synaptosomal fractions, including surface-localized neuronal receptors. However, co-expression of DISC1 normalized the reduction of synaptic protein levels. Furthermore, the overexpression of TDP-220C in neurons impaired induction of new protein synthesis in synaptosomal fractions by neuronal stimulation, which was also restored by co-expression of DISC1. These rescue effects were not caused simply by reduction in TDP-220C protein levels due to the co-expression of DISC1. These results indicate that the loss of functional DISC1 mediates the impairment of local translation in the neurons containing TDP-220C aggregates. Together, our findings suggest that co-aggregation of DISC1 with TDP-220C selectively perturbs the role of DISC1 in local translation in dendrites.
18.2.4 DISC1-Dependent Behavioral Impairment and Rescue in TDP-220C Mice
We examined in vivo behavioral consequences of the co-aggregation of DISC1 with TDP-220C. To examine this issue, we stereotaxically injected adeno-associated virus (AAV) encoding N-terminally Venus tagged TDP-220C into the frontal cortex of mice (hereafter termed TDP-220C mice) at 6 weeks old. The AAV infection resulted in formation of highly phosphorylated TDP-220C aggregates in neurons of frontal cortex. We observed endogenous DISC1 and full-length TDP-43 sequestered into the TDP-220C aggregates. Protein levels of synaptic genes were significantly reduced in TDP-220C mice, consistent with the results from cultured neurons.
In this study, we demonstrate that TDP-43, the causal protein in FTLD, co-aggregates with DISC1, a biological mediator for major mental illnesses, resulting in impaired local dendritic translation and disturbed social behaviors. The TDP-43-DISC1 co-aggregation compromised activity-dependent local dendritic translation and elicited mental deficits in FTLD model mice. Importantly, the psychiatric behaviors in the disease model mice were normalized by exogenous expression of DISC1 in frontal cortex, demonstrating that the loss of soluble, functional DISC1 due to co-aggregation is causally responsible for disturbed mental conditions in FTLD model mice. It has remained unclear why abnormal mental disorders are frequently observed in many neurodegenerative disorders. Given that local translation in dendrites is a critical process in higher brain functions (Buffington et al. 2014), our study has solved the long-standing puzzle of this neurological/psychiatric phenotypic overlap.
DISC1 was previously proposed to regulate synaptic plasticity and mental conditions, but the underlying molecular mechanisms have remained unclear (Tsuboi et al. 2015; Hayashi-Takagi et al. 2010). Our study reveals that DISC plays an unexpected role in determining activity-dependent local translation in dendrites, suggesting that DISC1 regulates the expression and hence function of postsynaptic genes in dendrites. Thus, our finding provides a candidate molecular basis for impaired synaptic plasticity and mental conditions observed in DISC1-knockdown neurons and DISC1 mutant mice (Hayashi-Takagi et al. 2010; Kvajo et al. 2011; Kuroda et al. 2011). Our results show that DISC1 regulates translation of mRNAs of critical synaptic genes, some of which are associated with psychiatric disorders. Our study validated, for TDP-43 and DISC1, the novel hypothesis that the co-aggregation of risk molecules for psychiatric diseases may account for latent psychiatric symptoms in neurodegenerative disorders.
We thank Akira Sawa, Yoko Machida, Yusuke Komi, Shigeo Murayama, Masaki Takao, Hiroyasu Akatsu, Koko Ishizuka, Miles Houslay and the members of Tanaka Laboratory for helpful discussion. We are grateful to the Brain Bank for Aging Research at Tokyo Metropolitan Institute of Gerontology and Fukushimura Brain Bank, and the families of the patients for providing brain samples used in this study. DNA sequencing and preparation of polyclonal antibodies were performed at the Research Resource Center facility in RIKEN Brain Science Institute. Animals were maintained by RIKEN Brain Science Institute animal facility. This work was supported by The Uehara Memorial Foundation (M.T.), Grant-in-Aid for Scientific Research on Innovative Areas from Ministry of Education, Culture, Sports, Science and Technology, Japan (26116004 to M.T.) and RIKEN Aging Project (MT).
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