Escalation on Kihon Checklist Scores Preceding the Certification of Long-Term Care Need in the Older Population in Japan. A 9-Year Retrospective Study.

Objectives: The Kihon Checklist (KCL) is valuable for predicting long-term care (LTC) certification. However, the precise association between KCL scores and the temporal dynamics of LTC need certification remains unclear. This study clarified the characteristic trajectory of KCL scores in individuals certified for LTC need. Methods: The KCL scores spanning from 2011 to 2019 were obtained from 5630 older individuals, including those certified for LTC need in November 2020, in Iiyama City, Nagano, Japan. We analyzed the KCL score trajectories using a linear mixed model, both before and after propensity score matching. Results: Throughout the 9-year observation period, the KCL scores consistently remained higher in the certified group compared to the non-certified group. Notably, a significant score increase occurred within the 3 years preceding LTC certification. Discussion: Our findings highlight the effectiveness of continuous surveillance using the KCL in identifying individuals likely to require LTC within a few years.

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Promotes Adult Neurogenesis and Neuronal Responsiveness in the Dentate Gyrus.

Motopsin, a serine protease encoded by PRSS12, is secreted by neuronal cells into the synaptic clefts in an activity-dependent manner, where it induces synaptogenesis by modulating Na+/K+-ATPase activity. In humans, motopsin deficiency leads to severe intellectual disability and, in mice, it disturbs spatial memory and social behavior. In this study, we investigated mice that overexpressed motopsin in the forebrain using the Tet-Off system (DTG-OE mice). The elevated agrin cleavage or the reduced Na+/K+-ATPase activity was not detected. However, motopsin overexpression led to a reduction in spine density in hippocampal CA1 basal dendrites. While motopsin overexpression decreased the ratio of mature mushroom spines in the DG, it increased the ratio of immature thin spines in CA1 apical dendrites. Female DTG-OE mice showed elevated locomotor activity in their home cages. DTG-OE mice showed aberrant behaviors, such as delayed latency to the target hole in the Barnes maze test and prolonged duration of sniffing objects in the novel object recognition test (NOR), although they retained memory comparable to that of TRE-motopsin littermates, which normally express motopsin. After NOR, c-Fos-positive cells increased in the dentate gyrus (DG) of DTG-OE mice compared with that of DTG-SO littermates, in which motopsin overexpression was suppressed by the administration of doxycycline, and TRE-motopsin littermates. Notably, the numbers of doublecortin- and 5-bromo-2'-deoxyuridine-labeled cells significantly increased in the DG of DTG-OE mice, suggesting increased adult neurogenesis. Importantly, our results revealed a new function in addition to modulating neuronal responsiveness and spine morphology in the DG: the regulation of neurogenesis.

Machine learning-based segmentation of the rodent hippocampal CA2 area from Nissl-stained sections.

The hippocampus is a center of learning, memory, and spatial navigation. This region is divided into the CA1, CA2, and CA3 areas, which are anatomically different from each other. Among these divisions, the CA2 area is unique in terms of functional relevance to sociality. The CA2 area is often manually detected based on the size, shape, and density of neurons in the hippocampal pyramidal cell layer, but this manual segmentation relying on cytoarchitecture is impractical to apply to a large number of samples and dependent on experimenters' proficiency. Moreover, the CA2 area has been defined based on expression pattern of molecular marker proteins, but it generally takes days to complete immunostaining for such proteins. Thus, we asked whether the CA2 area can be systematically segmented based on cytoarchitecture alone. Since the expression pattern of regulator of G-protein signaling 14 (RGS14) signifies the CA2 area, we visualized the CA2 area in the mouse hippocampus by RGS14-immunostaining and Nissl-counterstaining and manually delineated the CA2 area. We then established "CAseg," a machine learning-based automated algorithm to segment the CA2 area with the F1-score of approximately 0.8 solely from Nissl-counterstained images that visualized cytoarchitecture. CAseg was extended to the segmentation of the prairie vole CA2 area, which raises the possibility that the use of this algorithm can be expanded to other species. Thus, CAseg will be beneficial for investigating unique properties of the hippocampal CA2 area.

Conditioned medium from BV2 microglial cells having polyleucine specifically alters startle response in mice.

Repeat-associated non-AUG translation (RAN translation) is observed in transcripts that are causative for polyglutamine (polyQ) diseases and generates proteins with mono amino acid tracts such as polyalanine (polyA), polyleucine (polyL) and polyserine (polyS) in neurons, astrocytes and microglia. We have previously shown that microglia with aggregated polyQ led to defective differentiation and degeneration of neuron-like cells. However, it has not been determined whether only microglia containing a specific RAN product, but not other RAN products, is harmful in vitro and in vivo. Here we show that polyL-incorporating microglia specifically led to altered startle response in mice. Aggregated polyA, polyS and polyL induced aberrant differentiation of microglia-like BV2 cells. Differentiated PC12 cells treated with conditioned medium (CM) of polyS- and polyL- but not polyA-incorporating microglia-like BV2 cells showed retraction of neurites and loss of branch of neurites. Injection of the polyL-CM, but not polyA-CM and polyS-CM, into the lateral ventricle lowered startle response in mice. Consistently, polyL induced the highest expression of CD68 in BV2 cells. The lowered startle response was replicated in mice given the polyL-CM in the caudal pontine reticular nucleus (PnC), the key region of startle response. Thus, endogenous RAN proteins having polyL derived from polyQ diseases-causative genes in microglia might specifically impair startle response.

Differential brain expression pattern of Sez6 alternative splicing isoform with deleted transmembrane domain.

Seizure-related gene 6 (Sez6) is a transmembrane protein specifically localized on neuronal dendrites and responsible for dendritic branching and synapse formation. Alternative splicing produces three isoforms of Sez6 mRNAs: the dominant isoform encodes a transmembrane-type protein, whereas the two recessive isoforms encode transmembrane and secretory proteins. In the present study, to clarify the differential functions of these isoforms, the expression patterns resulting from Sez6 splicing isoforms were investigated in the mouse brain as well as in cultured neurons. The whole brains were sliced into coronal sections of 1-mm thickness, and brain areas were punched out from these coronal sections. The mRNA levels of each Sez6 isoform in the prefrontal cortex, cingulate cortex, striatum, hippocampus, and amygdala, where Sez6 expression has been reported previously, were analyzed using a qPCR technique, and primary neurons cultured under different treatment conditions were assessed in terms of increased Sez6 gene expression. Our results show that the splicing patterns of Sez6 were modulated in a brain area-specific manner. In particular, the striatum showed a characteristic splicing pattern of recessive isoforms. Moreover, neuronal activation by convulsant drug stimulation increased recessive isoforms like the dominant isoform in cultured cortical neurons at 5 or 10 days in vitro. In conclusion, alternative splicing of Sez6, as well as of other proteins expressed specifically in the brain, results in brain area-specific expression patterns. Furthermore, the alternative splicing of Sez6 may be modulated by drugs that elevate Sez6 gene expression.

Hemodynamic changes in the right ventrolateral prefrontal cortex relate to the psychological mood profile.

Previous reports indicate that the right ventrolateral prefrontal cortex (VLPFC) is involved in emotional regulation. However, most such studies were performed under unphysiological conditions, like the administration of transcranial direct current or repetitive transcranial magnetic stimulation. We have shown that the right VLPFC is stimulated by an acute bout of daily activity, such as cleaning. Here, we investigated the relationship between the right VLPFC and mood changes using this system. Fourteen young adults vacuumed a floor as a cleaning task and kept a standing position as a control task on separate days. The oxyhemoglobin (oxy-Hb) and deoxy-hemoglobin (deoxy-Hb) signals of the prefrontal cortex were measured during the tasks. The mood scale scores of Profile of Mood States 2nd edition (POMS) and Two-Dimensional Mood Scale (TDMS) were measured before and after both tasks. The differences in subscale scores between pre- and post-tasks in both scales were calculated as ΔPOMS and ΔTDMS. The cleaning task significantly increased the oxy-Hb signal in the bilateral VLPFC and right frontopolar, but did not affect the deoxy-Hb signals. The control task significantly decreased the oxy-Hb signal in some brain regions. The Confusion-Bewilderment score in POMS changed after the cleaning task. Importantly, the oxy-Hb signal in the right VLPFC was negatively correlated with the ΔPOMS Confusion-Bewilderment score. The activity of the right VLPFC stimulated by the cleaning task might have a correlation with the Confusion-Bewilderment mood state.

Exogenous polyserine and polyleucine are toxic to recipient cells.

Repeat-associated non-AUG (RAN) translation of mRNAs/transcripts responsible for polyglutamine (polyQ) diseases may generate peptides containing different mono amino acid tracts such as polyserine (polyS) and polyleucine (polyL). The propagation of aggregated polyQ from one cell to another is also an intriguing feature of polyQ proteins. However, whether the RAN translation-related polyS and polyL have the ability to propagate remains unclear, and if they do, whether the exogenous polyS and polyL exert toxicity on the recipient cells is also not known yet. In the present study, we found that aggregated polyS and polyL peptides spontaneously enter neuron-like cells and astrocytes in vitro. Aggregated polyS led to the degeneration of the differentiated neuron-like cultured cells. Likewise, the two types of aggregates taken up by astrocytes induced aberrant differentiation and cell death in vitro. Furthermore, injection of each of the two types of aggregates into the ventricles of adult mice resulted in their behavioral changes. The polyS-injected mice showed extensive vacuolar degeneration in the brain. Thus, the RAN translation-related proteins containing polyS and polyL have the potential to propagate and the proteins generated by all polyQ diseases might exert universal toxicity in the recipient cells.

Intracerebral Delivery of an OTR Antagonist and Subsequent Behavioral Testing in Prairie Voles.

Ethological analyses of rodents are indispensable in current neuroscience research. Unlike traditional experimental rodents, prairie voles (Microtus ochrogaster) are monogamous, preferring to huddle with their sociosexual partner rather than stranger voles. Partner preference in prairie voles is assessed using three transparent chambers. The right and left chambers are connected with the central chamber via hollow tubes. An opposite-sex partner vole is tethered to the right or left chamber, while a stranger vole is tethered to the other one. A subject prairie vole is placed in the central chamber and allowed to roam freely in the three chambers for 3 h. Behaviors are recorded in a video recorder placed in front of the chambers. The duration to huddle with the partner and stranger voles is hand-scored in a blinded fashion. In this partner preference test, acrylic chambers and other materials are custom-made. Here, we describe detailed specification of these apparatuses. In addition, we mention protocols for cannula implantation and intracerebroventricular injection as well as a standard protocol of a passive avoidance test in prairie voles.

Separation from a bonded partner alters neural response to inflammatory pain in monogamous rodents.

Pain experience is known to be modified by social factors, but the brain mechanisms remain unspecified. We recently established an animal model of social stress-induced hyperalgesia (SSIH) using a socially monogamous rodent, the prairie vole, in which males separated from their female partners (loss males) became anxious and displayed exacerbated inflammatory pain behaviors compared to males with partners (paired males). In the present study, to explore the neural pathways involved in SSIH, a difference in neuronal activation in pain-related brain regions, or "pain matrix", during inflammatory pain between paired and loss males was detected using Fos immunoreactivity (Fos-ir). Males were paired with a female and pair bonding was confirmed in all subjects using a partner preference test. During formalin-induced inflammatory pain, both paired and loss males showed a significant induction of Fos-ir throughout the analyzed pain matrix components compared to basal condition (without injection), and no group differences in immunoreactivity were found among the injected males in many brain regions. However, the loss males had significantly lower Fos-ir following inflammatory pain in the medial prefrontal cortex and nucleus accumbens shell than the paired males, even though base Fos-ir levels were comparable between groups. Notably, both regions with different Fos-ir are major components of the dopamine and oxytocin systems, which play critical roles in both pair bonding and pain regulation. The present results suggest the possibility that pain exacerbation by social stress emerges through alteration of signaling in social brain circuitry.

Motopsin deficiency imparts partial insensitivity to doxorubicin-induced hippocampal impairments in adult mice.

Motopsin is a serine protease that plays a crucial role in synaptic functions. Loss of motopsin function causes severe intellectual disability in humans. In this study, we evaluated the role of motopsin in the neuropathological development of cognitive impairments following chemotherapy, also known as chemobrain. Motopsin knockout (KO) and wild-type (WT) mice were intravenously injected with doxorubicin (Dox) or saline four times every 8 days and were evaluated for open field, novel object recognition, and passive avoidance tests. Parvalbumin-positive neurons in the hippocampus were immunohistochemically analyzed. Dox administration significantly decreased the total distance in the open field test in both WT and motopsin KO mice without affecting the duration spent in the center square. A significant interaction between the genotype and drug treatment was detected in the recognition index (the rate to investigate a novel object) in the novel object recognition test, although Dox treatment did not affect the total investigation time. Additionally, Dox treatment significantly decreased the recognition index in WT mice, whereas it tended to increase the recognition index in motopsin KO mice. Dox treatment did not affect the latency to enter a dark compartment in either WT or motopsin KO mice in the passive avoidance test. Interestingly, Dox treatment increased the parvalbumin-positive neurons in the stratum oriens of the hippocampus CA1 region of only WT mice, not motopsin KO mice. Our data suggest that motopsin deficiency imparted partial insensitivity to Dox-induced hippocampal impairments. Alternatively, motopsin may contribute to the neuropathology of chemobrain.

Oxytocin receptor antagonist reverses the blunting effect of pair bonding on fear learning in monogamous prairie voles.

Social relationships among spouses, family members, and friends are known to affect physical and mental health. In particular, long-lasting bonds between socio-sexual partners have profound effects on cognitive, social, emotional, and physical well-being. We have previously reported that pair bonding in monogamous prairie voles (Microtus ochrogaster) is prevented by a single prolonged stress (SPS) paradigm, which causes behavioral and endocrine symptoms resembling post-traumatic stress disorder (PTSD) patients in rats (Arai et al., 2016). Since fear memory function is crucial for anxiety-related disorders such as PTSD, we investigated the effects of pair bonding on fear learning in prairie voles. We applied an SPS paradigm to male prairie voles after the cohabitation with a male (cage-mate group) or female (pair-bonded group). The cage-mate group, but not the pair-bonded group, showed enhanced fear response in a contextual fear conditioning test following the SPS treatment. Immunohistochemical analyses revealed that cFos-positive cells in the central amygdala were increased in the pair-bonded group after the contextual fear conditioning test and that oxytocin immunoreactivity in the paraventricular nucleus of the hypothalamus was significantly higher in the pair-bonded group than the cage-mate group. This pair-bonding dependent blunting of fear memory response was confirmed by a passive avoidance test, another fear-based learning test. Interestingly, intracerebroventricular injection of an oxytocin receptor antagonist 30 min before the passive avoidance test blocked the blunting effect of pair bonding on fear learning. Thus, pair bonding between socio-sexual partners results in social buffering in the absence of the partner, blunting fear learning, which may be mediated by oxytocin signaling.

Vulnerability or resilience of motopsin knockout mice to maternal separation stress depending on adulthood behaviors.

BACKGROUND: Both environmental and genetic conditions contribute to the robust development of neuronal circuits and adulthood behaviors. Loss of motopsin gene function causes severe intellectual disability in humans and enhanced social behavior in mice. Furthermore, childhood maltreatment is a risk factor for some psychiatric disorders, and children with disabilities have a higher risk of abuse than healthy children. MATERIALS AND METHODS: In this study, we investigated the effects of maternal separation (MS) on adulthood behaviors of motopsin knockout (KO) and wild-type (WT) mice. RESULTS: The MS paradigm decreased the duration that WT mice stayed in the center area of an open field, but not for motopsin KO mice; however, it decreased the novel object recognition index in both genotypes. In the marble burying test, motopsin KO mice buried fewer marbles than WT mice, regardless of the rearing conditions. The MS paradigm slightly increased and reduced open arm entry in the elevated plus maze by WT and motopsin KO mice, respectively. In the three-chamber test, the rate of sniffing the animal cage was increased by the MS paradigm only for motopsin KO mice. After the three-chamber test, motopsin KO mice had fewer cFos-positive cells in the prelimbic cortex, which is involved in emotional response, than WT mice. In the infralimbic cortex, the MS paradigm decreased the number of cFos-positive cells in motopsin KO mice. CONCLUSION: Our results suggest that motopsin deficiency and childhood adversity independently affect some behaviors, but they may interfere with each other for other behaviors. Defective neuronal circuits in the prefrontal cortex may add to this complexity.

An acute bout of housework activities has beneficial effects on executive function.

PURPOSE: Although acute bouts of exercise reportedly have beneficial effects on executive function, inactive people may find it difficult to start exercising. In this study, we focused on housework activities (HAs) that generate a sense of accomplishment and require a mild intensity of physical activity. We examined the impact of an acute bout of HA on executive function and oxygenated hemoglobin (oxy-Hb) flow to related cortical regions. MATERIALS AND METHODS: Twenty-five participants (age, 18-21 years; mean, 19.88±0.60 years; six males and 19 females) underwent two experiments, ie, HA and control experiments, which were conducted on different days. Participants vacuumed a dirty floor in the HA experiment and mimicked the same motion with an unplugged vacuum cleaner on a clean floor in the control experiment. RESULTS: Heart rate recorded during the experiments showed no significant difference in the intensity of physical activity between control and HA groups. A questionnaire revealed a sense of accomplishment after completing the HA experiment. Participants performed the Stroop color-word task (SCWT) pre- and post-experiments; cortical hemodynamic changes were simultaneously monitored using functional near-infrared spectroscopy. Variation in Stroop interference scores for SCWT total response between pre- and post-experiments was signifi-cantly higher in the HA group than in the control group, and that for SCWT correct response showed a similar trend. Variation in the Stroop interference score for oxy-Hb flow to the right ventrolateral prefrontal cortex (R-VLPFC) showed the same trend. CONCLUSION: Thus, HAs may have a greater beneficial effect on executive function than other physical activities through the activation of PFC, including R-VLPFC.

Partner Loss in Monogamous Rodents: Modulation of Pain and Emotional Behavior in Male Prairie Voles.

OBJECTIVE: Pain is modulated by psychosocial factors, and social stress-induced hyperalgesia is a common clinical symptom in pain disorders. To provide a new animal model for studying social modulation of pain, we examined pain behaviors in monogamous prairie voles experiencing partner loss. METHODS: After cohabitation with novel females, males (n = 79) were divided into two groups on the basis of preference test scores. Half of the males of each group were separated from their partner (loss group), whereas the other half remained paired (paired group). Thus, males from both groups experienced social isolation. Open field tests, plantar tests, and formalin tests were then conducted on males to assess anxiety and pain-related behaviors. RESULTS: Loss males showing partner preferences (n = 20) displayed a significant increase in anxiety-related behavior in the open-field test (central area/total distance: 13.65% [1.58%] for paired versus 6.45% [0.87%] for loss; p < .001), a low threshold of thermal stimulus in the plantar test (withdrawal latencies: 9.69 [0.98] seconds for paired versus 6.15 [0.75] seconds for loss; p = .037), and exacerbated pain behaviors in the formalin test (total number of lifts: 40.33 [4.46] for paired versus 54.42 [1.91] for loss; p = .042) as compared with paired males (n = 20). Thermal thresholds in the plantar test significantly correlated with anxiety-related behavior in the open-field test (r = 0.64). No such differences were observed in the males that did not display partner preferences (r = 0.15). CONCLUSIONS: Results indicate that social bonds and their disruption, but not social housing without bonding followed by isolation, modulate pain and emotion in male prairie voles. The prairie vole is a useful model for exploring the neural mechanisms by which social relationships contribute to pain and nociceptive processing in humans.

A single prolonged stress paradigm produces enduring impairments in social bonding in monogamous prairie voles.

Traumatic events such as natural disasters, violent crimes, tragic accidents, and war, can have devastating impacts on social relationships, including marital partnerships. We developed a single prolonged stress (SPS) paradigm, which consisted of restraint, forced swimming, and ether anesthesia, to establish an animal model relevant to post-traumatic stress disorder. We applied a SPS paradigm to a monogamous rodent, the prairie vole (Microtus ochrogaster) in order to determine whether a traumatic event affects the establishment of pair bonds. We did not detect effects of the SPS treatment on anhedonic or anxiety-like behavior. Sham-treated male voles huddled with their partner females, following a 6day cohabitation, for a longer duration than with a novel female, indicative of a pair bond. In contrast, SPS-treated voles indiscriminately huddled with the novel and partner females. Interestingly, the impairment of pair bonding was rescued by oral administration of paroxetine, a selective serotonin reuptake inhibitor (SSRI), after the SPS treatment. Immunohistochemical analyses revealed that oxytocin immunoreactivity (IR) was significantly decreased in the supraoptic nucleus (SON), but not in the paraventricular nucleus (PVN), 7days after SPS treatment, and recovered 14days after SPS treatment. After the presentation of a partner female, oxytocin neurons labeled with Fos IR was significantly increased in SPS-treated voles compared with sham-treated voles regardless of paroxetine administration. Our results suggest that traumatic events disturb the formation of pair bond possibly through an interaction with the serotonergic system, and that SSRIs are candidates for the treatment of social problems caused by traumatic events. Further, a vole SPS model may be useful for understanding mechanisms underlying the impairment of social bonding by traumatic events.

N-Glycosylation modulates filopodia-like protrusions induced by sez-6 through regulating the distribution of this protein on the cell surface.

Seizure-related gene 6 (sez-6) is a trans-membrane protein expressed by neuronal cells that modulates dendritic branching. It has three clusters of eleven possible N-glycosylation sites in the extracellular domain region: sugar chain (SC)1-3, SC4-7, and SC8-11. Recent reports suggest that N-glycosylation modulates the membrane trafficking and function of trans-membrane proteins. Here, we studied the role of N-glycosylation in sez-6 function. We transfected mutants lacking one, two, or all N-glycosylation clusters into neuro2a cells. A mutant lacking all N-glycosylation was transported to the cell membrane. Mutants lacking one cluster (sez-6 ΔSC1-3, ΔSC4-7, ΔSC8-11) were evenly distributed on the cell membrane and secreted into the conditioned medium, as in wild-type sez-6; in contrast, the unglycosylated mutant, sez-6 ΔSC1-11, and mutants having only one cluster (sez-6 SC1-3, SC8-11) were localized in some portions on the cell membrane. Despite sez-6 SC4-7 having only one cluster, it was transported like the wild type. Among mutants behaving like the wild type, sez-6 ΔSC1-3 and ΔSC4-7 reduced neurite formation. Interestingly, mutants lacking SC4-7 (sez-6 ΔSC4-7) did not affect the formation of filopodia-like protrusions. In contrast, other mutants as well as the wild type induced it, suggesting that SC4-7 is crucial for filopodia-like protrusions. Our results indicate that N-glycosylation regulates cell morphology through modulating the cell surface distribution of sez-6 protein.

Tissue plasminogen activator modulates emotion in a social context.

Tissue plasminogen activator (tPA) is known to play physiologically and pathologically crucial roles in the central nervous system. However, it is still obscure whether it affects social behavior. We investigated social behavior and neuronal activation after social stimulation in tPA knockout (KO) mice. In a resident-intruder test, the latency to the first contact was significantly delayed in tPA KO mice compared with that in tPA heterogenic (Het) mice. However, tPA KO mice spent significantly more time undertaking active behavior than tPA Het mice did. In a sociability test, tPA KO mice significantly spent more time and walked a greater distance in the chamber containing an empty cage than tPA Het mice. Furthermore, tPA KO mice approached an empty cage more frequently than tPA Het mice did. In a social novelty test, there was no difference in the duration spent sniffing a stimulator mouse between genotypes. However, tPA KO mice approached even a familiar mouse more frequently than tPA Het mice did. tPA KO mice spent similar durations in a chamber containing a familiar mouse and that containing an unfamiliar mouse, and tPA KO mice walked a significantly greater distance in the former chamber than tPA Het mice did. Furthermore, at the cingulate and prelimbic cortices, the number of cFos-positive cells was significantly increased in tPA KO mice compared with that in tPA Het mice after social stimulation. Our results suggest that tPA modulates emotion in a social context through the function of the prefrontal cortex.

Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

Dermokine-ß impairs ERK signaling through direct binding to GRP78.

Dermokine-ß is abundant in stratified epithelia and in differentiating cultured keratinocytes. In this study, we investigated the role of dermokine-ß in differentiation of keratinocytes. Treatment of keratinocytes or skin tumor cells with dermokine-ß attenuated phosphorylation of extracellular-signal-regulated kinase (ERK). Exposure of cells to dermokine-ß, as well as its carboxyl-terminus domain peptide, interrupted phosphorylation of ERK and stimulated dermokine gene expression. Inhibition of ERK signaling by its specific inhibitor also increased dermokine expression level. A combination of chemical cross-linking and immunoprecipitation, followed by proteomics analyses, identified glucose-regulated protein 78 (GRP78) as a dermokine-ß-associated protein. Blockage of GRP78 expression by a specific siRNA abrogated actions of dermokine-ß. These findings provide novel insights into the physiological significance of dermokine-ß in the epidermis.