Masaki Tanaka

p62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate.

Proteolytic systems and the aggresome pathway contribute to preventing accumulation of cytotoxic aggregation-prone proteins. Although polyubiquitylation is usually required for degradation or aggresome formation, several substrates are processed independently of ubiquitin through a poorly understood mechanism. Here, we found that p62/SQSTM1, a multifunctional adaptor protein, was involved in the selective autophagic clearance of a non-ubiquitylated substrate, namely an aggregation-prone isoform of STAT5A (STAT5A_ΔE18). By using a cell line that stably expressed STAT5A_ΔE18, we investigated the properties of its aggregation and degradation. We found that STAT5A_ΔE18 formed non-ubiquitylated aggresomes and/or aggregates by impairment of proteasome functioning or autophagy. Transport of these aggregates to the perinuclear region was inhibited by trichostatin A or tubacin, inhibitors of histone deacetylase (HDAC), indicating that the non-ubiquitylated aggregates of STAT5A_ΔE18 were sequestered into aggresomes in an HDAC6-dependent manner. Moreover, p62 was bound to STAT5A_ΔE18 through its PB1 domain, and the oligomerization of p62 was required for this interaction. In p62-knockdown experiments, we found that p62 was required for autophagic clearance of STAT5A_ΔE18 but not for its aggregate formation, suggesting that the binding of p62 to non-ubiquitylated substrates might trigger their autophagic clearance.

Relaxin-3-Deficient Mice Showed Slight Alteration in Anxiety-Related Behavior

Relaxin-3 is a neuropeptide belonging to the relaxin/insulin superfamily. Studies using rodents have revealed that relaxin-3 is predominantly expressed in neurons in the nucleus incertus (NI) of the pons, the axons of which project to forebrain regions including the hypothalamus. There is evidence that relaxin-3 is involved in several functions, including food intake and stress responses. In the present study, we generated relaxin-3 gene knockout (KO) mice and examined them using a range of behavioral tests of sensory/motor functions and emotion-related behaviors. The results revealed that relaxin-3 KO mice exhibited normal growth and appearance, and were generally indistinguishable from wild genotype littermates. There was no difference in bodyweight among genotypes until at least 28 weeks after birth. In addition, there were no significant differences between wild-type and KO mice in locomotor activity, social interaction, hot plate test performance, fear conditioning, depression-like behavior, and Y-maze test performance. However, in the elevated plus maze test, KO mice exhibited a robust increase in the tendency to enter open arms, although they exhibited normal performance in a light/dark transition test and showed no difference from wild-type mice in the time spent in central area in the open field test. On the other hand, a significant increase in the acoustic startle response was observed in KO mice. These results indicate that relaxin-3 is slightly involved in the anxiety-related behavior.

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson’s disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.

Relaxin-3⁄insulin-like peptide 7, a neuropeptide involved in the stress response and food intake

Relaxin‐3, also known as insulin‐like peptide‐7, is a newly‐identified peptide of the insulin superfamily. All members of this superfamily have a similar structure, which consists of two subunits (A‐chain and B‐chain) linked by disulfide bonds. Relaxin‐3 is so named because it has a motif that can interact with the relaxin receptor. By contrast to other relaxins, relaxin‐3 is mainly expressed in the brain and testis. In rodent brain, anatomical studies have revealed its predominant expression in neurons of the nucleus incertus of the dorsal pons, and a few other regions of the brainstem. On the other hand, relaxin‐3‐expressing nerve fibers and the relaxin‐3 receptors, RXFP3 and RXFP1, are widely distributed in the forebrain, with the hypothalamus being one of the most densely‐innervated regions. Therefore, relaxin‐3 is considered to exert various actions through its ligand‐receptor system. This minireview describes the expression of relaxin‐3 in the brain, as well as its functions in the hypothalamus, including the stress response and food intake.

Differential Expression of Alpha-Synuclein in Hippocampal Neurons

α-Synuclein is the major pathological component of synucleinopathies including Parkinsons disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.

Serotonin2C receptors in the nucleus accumbens are involved in enhanced alcohol-drinking behavior.

Dopamine and serotonin (5‐HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol‐drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol‐exposed mice, the expression of 5‐HT2C receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5‐HT2C receptor significantly increased in the ACC. The expression of 5‐HT7 receptor mRNA increased in the ACC and DRN. Contents of 5‐HT decreased in the ACC shell (ACCS) and DRN of the alcohol‐exposed mice. The basal extracellular releases of dopamine (DA) and 5‐HT in the ACCS increased more in the alcohol‐exposed mice than in alcohol‐naïve mice. The magnitude of the alcohol‐induced ACCS DA and 5‐HT release in the alcohol‐exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACCS of the 5‐HT2C receptor antagonist, SB‐242084, suppressed voluntary alcohol‐drinking behavior in the alcohol‐exposed mice. But the i.p. administration of the 5‐HT7 receptor antagonist, SB‐258719, did not have significant effects on alcohol‐drinking behavior in the alcohol‐exposed mice. The effects of the 5‐HT2C receptor antagonist were not observed in the air‐exposed control mice. These results suggest that adaptations of the 5‐HT system, especially the upregulation of 5‐HT2C receptors in the ACCS, are involved in the development of enhanced voluntary alcohol‐drinking behavior.

Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinsons disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.

Brain region-dependent differential expression of alpha-synuclein

α‐Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α‐synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinsons disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α‐synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α‐synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α‐synuclein was highly expressed in the neuronal cell bodies of some early PD‐affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α‐synuclein was mostly accompanied by expression of vesicular glutamate transporter‐1, an excitatory presynaptic marker. In contrast, expression of α‐synuclein in the GABAergic inhibitory synapses was different among brain regions. α‐Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD‐affected human brain regions express high levels of perikaryal α‐synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α‐synuclein are different in various brain regions. J. Comp. Neurol. 524:1236–1258, 2016.

HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis

ABSTRACT Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.

Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors

Serotonin 2C receptors (5-HT(2C)R) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT(2C)R mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT(2C)R mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT(2C)R, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT(2C)R mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT(2C)R mRNA editing underlie alcohol preference in mice.