Division of labor among H3K4 Methyltransferases Defines Distinct Facets of Homeostatic Plasticity.

Heterozygous mutations in any of the six H3K4 methyltransferases (KMT2s) result in monogenic neurodevelopmental disorders, indicating nonredundant yet poorly understood roles of this enzyme family in neurodevelopment. Recent evidence suggests that histone methyltransferase activity may not be central to KMT2 functions; however, the enzymatic activity is evolutionarily conserved, implicating the presence of selective pressure to maintain the catalytic activity. Here, we show that H3K4 methylation is dynamically regulated during prolonged alteration of neuronal activity. The perturbation of H3K4me by the H3.3K4M mutant blocks synaptic scaling, a form of homeostatic plasticity that buffers the impact of prolonged reductions or increases in network activity. Unexpectedly, we found that the six individual enzymes are all necessary for synaptic scaling and that the roles of KMT2 enzymes segregate into evolutionary-defined subfamilies: KMT2A and KMT2B (fly-Trx homologs) for synaptic downscaling, KMT2C and KMT2D (Trr homologs) for upscaling, and KMT2F and KMT2G (dSet homologs) for both directions. Selective blocking of KMT2A enzymatic activity by a small molecule and targeted disruption of the enzymatic domain both blocked the synaptic downscaling and interfered with the activity-dependent transcriptional program. Furthermore, our study revealed specific phases of synaptic downscaling, i.e., induction and maintenance, in which KMT2A and KMT2B play distinct roles. These results suggest that mammalian brains have co-opted intricate H3K4me installation to achieve stability of the expanding neuronal circuits.

The five-item modified frailty index predicts long-term outcomes in elderly patients undergoing colorectal cancer surgery.

BACKGROUND: Frailty has been globally recognized as a predictor of adverse postoperative outcomes. Frailty assessment using the five-factor modified frailty index (5-mFI) has recently gained traction; however, long-term outcomes are unknown in colorectal cancer (CRC) surgery. This study aimed to investigate whether the 5-mFI predicted long-term survival and cause of death on the basis of frailty severity in elderly patients who underwent CRC surgery and to determine the risk factors for mortality. METHODS: A total of 299 patients underwent CRC surgery with curative intent between January 2013 and December 2017. Patients were divided into three groups by the 5-mFI score: group 1 (5-mFI: 0 or 1; n = 164): no frailty; group 2 (5-mFI: 2; n = 91): moderate frailty; and group 3 (5-mFI: ≥ 3; n = 44): severe frailty. Clinicopathological variables, namely comorbidities, 5-mFI, prognostic nutrition index, operative/postoperative data, and outcome, including cause of death, were compared between the three groups. To identify factors associated with death from CRC- and non-CRC-related causes, univariate and multivariate analyses using a Cox regression model were performed. RESULTS: The immediate postoperative morbidity of patients with Clavien-Dindo grade ≥ III complications (9.1%) in group 3 was not significantly different from that in group 1 (9.1%) or group 2 (14.3%); however, the 30-day mortality rate (4.5%) in group 3 was significantly higher. Long-term disease-free survival was similar between frailty groups, suggesting that CRC surgery provides oncological benefit to patients irrespective of frailty. The 5-year survival rates in groups 1, 2, and 3 were 83.5%, 71.2%, and 47.9%, respectively, showing a significantly lower survival rate as frailty advanced. Sixty percent of the deaths in frail patients were due to respiratory failure and cardiovascular diseases. Multivariate analysis demonstrated that advanced age, higher 5-mFI score, and longer postoperative hospital stay were risk factors for mortality unrelated to CRC. Multivariate analysis also revealed that advanced tumor stage, carcinoembryonic antigen ≥ 5 ng/ml, undifferentiated tumor, and R1 resection were risk factors for CRC-related mortality. CONCLUSIONS: The 5-mFI score can predict postoperative short- and long-term outcomes and risk factors for mortality unrelated to CRC. Additionally, long-term survival was negatively associated with the 5-mFI score.

Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to the plasma membrane either via the SNX27-Retromer-WASH pathway or via the recently discovered SNX17-Retriever-CCC-WASH pathway. While SNX27 is responsible for the recycling of key neuronal receptors, the roles of SNX17 in neurons are less understood. Here, using cultured hippocampal neurons, we demonstrate that the SNX17 pathway regulates synaptic function and plasticity. Disruption of this pathway results in a loss of excitatory synapses and prevents structural plasticity during chemical long-term potentiation (cLTP). cLTP drives SNX17 recruitment to synapses, where its roles are in part mediated by regulating the surface expression of ß1-integrin. SNX17 recruitment relies on NMDAR activation, CaMKII signaling, and requires binding to the Retriever and PI(3)P. Together, these findings provide molecular insights into the regulation of SNX17 at synapses and define key roles for SNX17 in synaptic maintenance and in regulating enduring forms of synaptic plasticity.

RAI1 Regulates Activity-Dependent Nascent Transcription and Synaptic Scaling.

Long-lasting forms of synaptic plasticity such as synaptic scaling are critically dependent on transcription. Activity-dependent transcriptional dynamics in neurons, however, remain incompletely characterized because most previous efforts relied on measurement of steady-state mRNAs. Here, we use nascent RNA sequencing to profile transcriptional dynamics of primary neuron cultures undergoing network activity shifts. We find pervasive transcriptional changes, in which ∼45% of expressed genes respond to network activity shifts. We further link retinoic acid-induced 1 (RAI1), the Smith-Magenis syndrome gene, to the transcriptional program driven by reduced network activity. Remarkable agreement among nascent transcriptomes, dynamic chromatin occupancy of RAI1, and electrophysiological properties of Rai1-deficient neurons demonstrates the essential roles of RAI1 in suppressing synaptic upscaling in the naive network, while promoting upscaling triggered by activity silencing. These results highlight the utility of bona fide transcription profiling to discover mechanisms of activity-dependent chromatin remodeling that underlie normal and pathological synaptic plasticity.

Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.

The excitatory-to-inhibitory functional switch of γ-aminobutyric acid (GABA; GABA switch), which normally occurs in the first to the second postnatal week in the hippocampus, is necessary for the development of appropriate central nervous system function. A deficit in GABAergic inhibitory function could cause excitatory/inhibitory (E/I) neuron imbalance that is found in many neurodegenerative disorders. In the present study, we examined whether neonatal stress can affect the timing of the GABA functional switch and cause disorders during adolescence. Neonatal stress was induced in C57BL/6J male mouse pups by maternal separation (MS) on postnatal days (PND) 1-21. Histological quantification of K+-Cl- co-transporter (KCC2) and Ca2+ imaging were performed to examine the timing of the GABA switch during the MS period. To evaluate the influence of neonatal MS on adolescent hippocampal function, we quantified KCC2 expression and evaluated hippocampal-related behavioral tasks at PND35-38. We showed that MS delayed the timing of the GABA switch in the hippocampus and inhibited the increase in membrane KCC2 expression, with KCC2 expression inhibition persisting until adolescence. Behavioral tests showed impaired cognition, declined attention, hyperlocomotion, and aggressive character in maternally separated mice. Taken together, our results show that neonatal stress delayed the timing of the GABA switch, which could change the E/I balance and cause neurodegenerative disorders in later life.

Sensitivity of mitochondrial DNA depleted ρ0 cells to H2O2 depends on the plasma membrane status.

To clarify the relationship between mitochondrial DNA (mtDNA)-depleted ρ0 cells and the cellular sensitivity to hydrogen peroxide (H2O2), we established HeLa and SAS ρ0 cell lines and investigated their survival rate in H2O2, radical scavenging enzymes, plasma membrane potential status, and chronological change in intracellular H2O2 amount under the existence of extracellular hydrogen peroxide compared with the parental cells. The results revealed that ρ0 cells had higher sensitivity to H2O2 than their parental cells, even though the catalase activity of ρ0 cells was up-regulated, and the membrane potential of the ρ0 cells was lower than their parental cells. Furthermore, the internal H2O2 amount significantly increased only in ρ0 cells after 50 µM H2O2 treatment for 1 h. These results suggest that plasma membrane status of ρ0 cells may cause degradation, and the change could lead to enhanced membrane permeability to H2O2. As a consequence, ρ0 cells have a higher H2O2 sensitivity than the parental cells.

The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.

K(+)-Cl(-) co-transporter (KCC2) and Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2(ser940)) immunoreactivity. The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2(ser940), corresponding to that of KCC2. The data in this article support the results of a previous study [1] and provide the details of immunohistological methods. Interpretation of the data in this article can be found in "Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice" by Tsukahara et al. [1].

A relation between osteoclastogenesis inhibition and membrane-type estrogen receptor GPR30.

Disruption of the cooperative balance between osteoblasts and osteoclasts causes various bone disorders, some of which are because of abnormal osteoclast recruitment. Osteoporosis, one of the bone disorders, is not effectively treated by currently available medicines. In addition to the development of novel drugs for palliative treatment, the exploitation of novel compounds for preventive treatment is important in an aging society. Quercetin, a major flavonoid found in many fruits and vegetables, has been expected to inhibit cancer and prevent several diseases because of its anti-inflammatory and estrogenic functions. It has been reported that quercetin has the potential to reduce bone resorption, but the mechanism by which this compound affects the differentiation of osteoclasts remains unknown. Here, using a bone marrow cell-based in vitro osteoclast differentiation system from bone marrow cells, we found that the ability of quercetin to inhibit osteoclastogenesis was related to its estrogenic activity. The inhibition was partially blocked by a specific antagonist for the nuclear receptor estrogen receptor α, but a specific antagonist of the membrane-type receptor GPR30 completely ablated this inhibition. Furthermore, quercetin suppressed the transient increase of Akt phosphorylation induced by the stimulation of macrophage colony-stimulating factor and receptor activator of NF-κB ligand with no effect on MAPK phosphorylation, suggesting exquisite crosstalk between cytokine receptor and G-protein coupled receptor signaling. These results indicate the important role of GPR30 in osteoclast differentiation and provide new insights to the development of new treatments for osteoporosis.

Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice.

The balance of cation-chloride co-transporters, particularly KCC2 and NKCC1, is critical for GABAergic inhibitory signaling. However, KCC2/NKCC1 balance is disrupted in many neurodegenerative diseases. Moreover, correlations between chronic stress, KCC2 and NKCC1 in the hippocampus remain poorly understood. Despite the fact that emotional disorders in humans are far more prevalent in women, there have been relatively few studies about female subjects. Here we investigated behaviors and expression patterns of KCC2 and NKCC1 in the hippocampi of female mice under chronic stress. Repeated stress (RS) was induced in experimental mice by repeated forced water administration. Then, expression patterns of GABAergic signaling molecules were identified by immunohistochemical analysis and performance was assessed using several behavioral tests. The results of semi-quantitative analysis showed that RS decreased KCC2 expression and increased NKCC1 expression in membranes of granular and pyramidal cells in the hippocampus. The novel object recognition (NOR) test and sociability test revealed that RS induced cognitive and sociability deficits, whereas RS increased the time spent in the open arms of the elevated plus maze test and induced attention deficits in other tests. In summary, RS induced alterations in membrane KCC2/NKCC1 balance in the hippocampus of female mice, which may contribute to GABAergic disinhibition associated with cognitional, sociability and attention deficits.