Suppressor of Cytokine Signalling 2 (SOCS2) Regulates Numbers of Mature Newborn Adult Hippocampal Neurons and Their Dendritic Spine Maturation.

Overexpression of suppressor of cytokine signalling 2 (SOCS2) has been shown to promote hippocampal neurogenesis in vivo and promote neurite outgrowth of neurons in vitro. In the adult mouse brain, SOCS2 is most highly expressed in the hippocampal CA3 region and at lower levels in the dentate gyrus, an expression pattern that suggests a role in adult neurogenesis. Herein we examine generation of neuroblasts and their maturation into more mature neurons in SOCS2 null (SOCS2KO) mice. EdU was administered for 7 days to label proliferative neural precursor cells. The number of EdU-labelled doublecortin+ neuroblasts and NeuN+ mature neurons they generated was examined at day 8 and day 35, respectively. While no effect of SOCS2 deletion was observed in neuroblast generation, it reduced the numbers of EdU-labelled mature newborn neurons at 35 days. As SOCS2 regulates neurite outgrowth and dentate granule neurons project to the CA3 region, alterations in dendritic arborisation or spine formation may have correlated with the decreased numbers of EdU-labelled newborn neurons. SOCS2KO mice were crossed with Nes-CreERT2/mTmG mice, in which membrane eGFP is inducibly expressed in neural precursor cells and their progeny, and the dendrite and dendritic spine morphology of newborn neurons were examined at 35 days. SOCS2 deletion had no effect on total dendrite length, number of dendritic segments, number of branch points or total dendritic spine density but increased the number of mature "mushroom" spines. Our results suggest that endogenous SOCS2 regulates numbers of EdU-labelled mature newborn adult hippocampal neurons, possibly by mediating their survival and that this may be via a mechanism regulating dendritic spine maturation.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology.

Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet-On cells. Over-expression of SOCS2 in PC12 Tet-On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over-expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2-induced neurite outgrowth in PC12 Tet-On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.

Melatonin does not reduce delirium severity in hospitalized older adults: Results of a randomized placebo-controlled trial.

BACKGROUND: Delirium is common in older inpatients, causing distress, cognitive decline, and death. Current therapies are unsatisfactory, limited by lack of efficacy and adverse effects. There is an urgent need for effective delirium treatment. Sleep wake cycle is disturbed in delirium; endogenous Melatonin is perturbed, and exogenous Melatonin is a safe and effective medication for sleep disorders. This study aims to determine the effect of oral Melatonin 5 mg immediate release (IR) nightly for five nights on the severity of delirium in older (≥65 years) medical inpatients. METHODS: This was a double-blinded, randomized controlled trial in general internal medicine units of a tertiary teaching hospital. Older inpatients with Confusion Assessment Method positive, hyperactive or mixed delirium within 48 h of admission or onset of in-hospital delirium were included. The primary outcome was change in delirium severity measured with the Memorial Delirium Assessment Scale (MDAS). A previous pilot trial showed 120 participants randomized 1:1 to Melatonin or Placebo would provide 90% power to demonstrate a 3-point reduction in the MDAS. RESULTS: One hundred and twenty participants were randomized, 61 to Melatonin 5 mg and 59 to Placebo. The medication was well tolerated. The mean MDAS improvement was 4.9 (SD 7.6) in the Melatonin group and 5.4 (SD 7.2) in the Placebo group, p-value 0.42, a non-significant difference. A post-hoc analysis showed length of stay (LOS) was shorter in the intervention group (median 9 days [Interquartile Range (IQR) 4, 12] vs. Placebo group 10 [IQR 6, 16] p-value = 0.033, Wilcoxon Rank Sum test). CONCLUSIONS: This trial does not support the hypothesis that Melatonin reduces the severity of delirium. This may be due to no effect of Melatonin, a smaller effect than anticipated, an effect not captured on a multidimensional delirium assessment scale, or a type II statistical error. Melatonin may improve LOS; this hypothesis should be studied.

Nanodiamonds with silicon vacancy defects for nontoxic photostable fluorescent labeling of neural precursor cells.

Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiV-containing NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

Patterns of Objectively Measured Sedentary Behavior and Physical Activity and Their Association with Changes in Physical and Functional Performance in Geriatric Rehabilitation Inpatients.

OBJECTIVES: To examine whether The Ending PyJama (PJ) Paralysis campaign, focused on increasing in-hospital physical activity, affects objectively measured sedentary behavior and physical activity patterns and if these are associated with changes in physical and functional performance in geriatric rehabilitation inpatients. DESIGN: Quasi-experimental study. SETTING AND PARTICIPANTS: Within the REStORing health of acutely unwell adulTs (RESORT) observational, longitudinal cohort of geriatric rehabilitation inpatients, the Ending PJ Paralysis campaign was implemented on 2 out of 4 wards. METHODS: Objectively measured sedentary behavior and physical activity were measured by an inertial sensor (ActivPAL4) for 1 week, comparing control (non-PJ) and intervention (PJ) groups using linear mixed models. Mean sedentary behavior and physical activity measures and their association with physical and functional performance changes were investigated by linear regression analyses, stratified by low vs high performance at admission using the median as a cut-off. RESULTS: A total of 145 (n = 68 non-PJ and n = 77 PJ) inpatients with a mean age of 83.0 (7.7) years (55.9% female inpatients) were included. The median nonupright time was 23.1 [22.1-23.6] and 23.0 [21.8-23.6] hours/day for non-PJ and PJ groups, respectively. Objectively measured sedentary behavior and physical activity measures did not significantly change over measurement days and were independent of the Ending PJ Paralysis campaign. For inpatients with low performance at admission, lower sedentary behavior [B(SE) -0.013 (0.005) to -0.157 (0.045), P < .01] and higher physical activity [B(SE) 0.033 (0.007) to 0.814 (0.200), P < .01] measures were associated with improved physical performance. In addition, lower sedentary behaviour [B(SE) = -0.058 (0.024), P < .05 and higher physical activity [B (SE) 0.060 (0.024) to 0.683 (0.182), P < .05] were associated with improved instrumental functional performance. CONCLUSIONS AND IMPLICATIONS: In geriatric rehabilitation inpatients, the Ending PJ Paralysis campaign did not affect objectively measured sedentary behavior and physical activity patterns. Lower mean sedentary behaviour and higher physical activity measures were associated with improved physical and functional performance in inpatients with low performance.

The Rho kinase pathway regulates mouse adult neural precursor cell migration.

Adult neural precursor cells (NPCs) in the subventricular zone (SVZ) normally migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB). Following neural injury, they also migrate to the site of damage. This study investigated the role of Rho-dependent kinase (ROCK) on the migration of NPCs in vitro and in vivo. In vitro, using neurospheres or SVZ explants, inhibition of ROCK using Y27632 promoted cell body elongation, process protrusion, and migration, while inhibiting NPC chain formation. It had no effect on proliferation, apoptosis, or differentiation. Both isoforms of ROCK were involved. Using siRNA, knockdown of both ROCK1 and ROCK2 was required to promote NPC migration and morphological changes; knockdown of ROCK2 alone was partially effective, with little/no effect of knockdown of ROCK1 alone. In vivo, infusion of Y27632 plus Bromodeoxyuridine (BrdU) into the lateral ventricle for 1 week reduced the number of BrdU-labeled NPCs in the OB compared with BrdU infusion alone. However, ROCK inhibition did not affect the tangential-to-radial switch of NPC migration, as labeled cells were present in all OB layers. The decrease in NPC number at the OB was not attributed to a decrease in NPCs at the SVZ. However, ROCK inhibition decreased the density of BrdU-labeled cells in the RMS and increased the distribution of these cells to ectopic brain regions, such as the accessory olfactory nucleus, where the majority differentiated into neurons. These findings suggest that ROCK signaling regulates NPC migration via regulation of cell-cell contact and chain migration.

Determinants of instrumented sedentary and physical activity behavior in geriatric rehabilitation inpatients: RESORT.

BACKGROUND: Physical inactivity in hospitalized older adults is highly prevalent and associated with detrimental health outcomes. Understanding its determinants is important for prognosis and tailoring interventions in geriatric rehabilitation inpatients. METHODS: Within the REStORing health of acutely unwell adulTs (RESORT) observational, longitudinal cohort, geriatric rehabilitation inpatients wore an inertial sensor (ActivPAL4) for one week to objectively assess instrumented sedentary behavior (i-SB) and physical activity (i-PA). Determinants were grouped in five geriatric domains: morbidity, cognition/psychology, physical performance, functional performance, and nutritional status. Their association with i-SB (mean sitting, lying, non-upright time) and i-PA (mean number of steps, sit-to-stand transitions and upright time) quintiles were examined using multivariate ordinal logistic regression analyses with Bonferroni correction (p < 0.006). RESULTS: A total of 145 inpatients were included (mean age 83.0, SD 7.7 years; 55.9% females). More comorbidities were associated with a lower daily number of steps (OR:0.91, 95%CI: 0.86-0.96) and lower upright time (OR:0.93, 95%CI: 0.88-0.98). Depressive symptoms (higher Hospital Anxiety and Depression Scale score) were associated with higher non-upright time (OR: 1.12, 95%CI: 1.03-1.21) and lower upright time (OR: 0.89, 95%CI: 0.83-0.96). Better physical performance (higher Functional Ambulation Classification, gait speed, and Short Physical Performance Battery score) was associated with lower i-SB measures (OR range: 0.07-0.78, p < 0.0005) and higher i-PA measures (OR range: 1.35-19.50, p < 0.0005). Higher functional performance (Katz index of Activities of Daily Living score) was associated with lower i-SB measures (OR range: 0.61-0.69, p ≤ 0.003) and higher i-PA measures (OR range: 1.60-3.64, p < 0.0005). Being malnourished was associated with lower i-PA measures (OR range: 0.29-0.32, p ≤ 0.004). CONCLUSIONS: Worse morbidity, depressive symptoms, worse physical and functional performance, and worse nutritional status were associated with higher i-SB and lower i-PA. These determinants should be taken into account while designing and promoting multidisciplinary physical activity interventions.

Fibroblast growth factor-9 inhibits astrocyte differentiation of adult mouse neural progenitor cells.

Fibroblast growth factor-9 (FGF9) is expressed in the CNS and is reported to be a mitogen for glial cells, to promote neuronal survival, and to retard oligodendrocyte differentiation. Here we examined the effects of FGF9 on the differentiation, survival, and proliferation of adult neural progenitor cells derived from the adult mouse subventricular zone. FGF9 by itself induced neurosphere proliferation, but its effects were modest compared with those of epidermal growth factor and FGF2. When neurospheres were dissociated and plated for differentiation, FGF9 increased total cell number over time in a dose-dependent manner. Ki67 immunostaining and bromodeoxyuridine incorporation indicated that this was at least partially due to the continued presence of proliferative nestin-positive neural progenitor cells and betaIII tubulin-positive neuronal precursors. FGF9 also promoted cell survival as indicated by a decreased number of TUNEL-positive cells over time. Assessment of differentiation showed that FGF9 increased neuron generation that reflected the increase in total cell number; however, the percentage of progenitor cells differentiating into neurons was slightly decreased. FGF9 had a modest effect on oligodendrocyte generation, although it appeared to slow the maturation of oligodenrocytes at higher concentrations. The most marked effect on differentiation was an almost total lack of glial fibrillary acidic protein (GFAP)-positive astrocytes up to 7 days following FGF9 addition, indicating that astrocyte differentiation was strongly inhibited. Total inhibition required prolonged treatment, although a 1-hr pulse was sufficient for partial inhibition, and bone morphogenic protein-4 could partially overcome the FGF9 inhibition of astrocyte differentiation. FGF9 therefore has multiple effects on adult neural precursor cell function, enhancing neuronal precursor proliferation and specifically inhibiting GFAP expression.

Explant Methodology for Analyzing Neuroblast Migration.

The subventricular zone (SVZ) in the mammalian forebrain contains stem/progenitor cells that migrate through the rostral migratory stream (RMS) to the olfactory bulb throughout adulthood. SVZ-derived explant cultures provide a convenient method to assess factors regulating the intermediary stage of neural stem/progenitor cell migration. Here, we describe the isolation of SVZ-derived RMS explants from the neonatal mouse brain, and the conditions required to culture and evaluate their migration.

Suppressor of Cytokine Signaling-2 (SOCS2) Regulates the Microglial Response and Improves Functional Outcome after Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) is frequently characterized by neuronal, axonal and myelin loss, reactive gliosis and neuroinflammation, often associated with functional deficits. Endogenous repair mechanisms include production of new neurons from precursor cells, but usually the new neurons fail to integrate and survive more than a few weeks. This is in part mediated by the toxic and inflammatory environment present in the injured brain which activates precursor cells to proliferate and differentiate but limits survival of the newborn progeny. Therefore, an understanding of mechanisms that regulate production and survival of newborn neurons and the neuroinflammatory response after brain injury may lead to therapeutic options to improve outcomes. Suppressor of Cytokine Signaling 2 (SOCS2) promotes hippocampal neurogenesis and survival of newborn neurons in the adult brain and regulates anti-inflammatory responses in the periphery, suggesting it may be a useful candidate to improve outcomes of TBI. In this study the functional and cellular responses of SOCS2 over-expressing transgenic (SOCS2Tg) mice were compared to wildtype littermates following mild or moderately severe TBI. Unlike wildtype controls, SOCS2Tg mice showed functional improvement on a ladder test, with a smaller lesion volume at 7d post injury and increased numbers of proliferative CD11b+ microglia/macrophages at 35d post-injury in the mild injury paradigm. At 7d post-moderately severe injury there was an increase in the area covered by cells expressing an anti-inflammatory M2 phenotype marker (CD206+) but no difference in cells with a pro-inflammatory M1 phenotype marker (CD16/32+). No effect of SOCS2 overexpression was observed in production or survival of newborn neurons, even in the presence of the neuroprotective agent erythropoietin (EPO). Therefore, SOCS2 may improve outcome of TBI in mice by regulating aspects of the neuroinflammatory response, promoting a more anti-inflammatory environment, although this was not sufficient to enhance survival of newborn cortical neurons.

Rho kinase inhibition following traumatic brain injury in mice promotes functional improvement and acute neuron survival but has little effect on neurogenesis, glial responses or neuroinflammation.

Inhibition of the Rho/Rho kinase pathway has been shown to be beneficial in a variety of neural injuries and diseases. In this manuscript we investigate the role of Rho kinase inhibition in recovery from traumatic brain injury using a controlled cortical impact model in mice. Mice subjected to a moderately severe TBI were treated for 1 or 4 weeks with the Rho kinase inhibitor Y27632, and functional outcomes and neuronal and glial cell responses were analysed at 1, 7 and 35 days post-injury. We hypothesised that Y27632-treated mice would show functional improvement, with augmented recruitment of neuroblasts from the SVZ and enhanced survival of newborn neurons in the pericontusional cortex, with protection against neuronal degeneration, neuroinflammation and modulation of astrocyte reactivity and blood-brain-barrier permeability. While Rho kinase inhibition enhanced recovery of motor function after trauma, there were no substantial increases in the recruitment of DCX(+) neuroblasts or the number of BrdU(+) or EdU(+) labelled newborn neurons in the pericontusional cortex of Y27632-treated mice. Inhibition of Rho kinase significantly reduced the number of degenerating cortical neurons at 1day post-injury compared to saline controls but had no longer term effect on neuronal degeneration, with only modest effects on astrocytic reactivity and macrophage/microglial responses. Overall, this study showed that Rho kinase contributes to acute neurodegenerative processes in the injured cortex but does not play a significant role in SVZ neural precursor cell-derived adult neurogenesis, glial responses or blood-brain barrier permeability following a moderately severe brain injury.

EphrinB3 restricts endogenous neural stem cell migration after traumatic brain injury.

Traumatic brain injury (TBI) leads to a series of pathological events that can have profound influences on motor, sensory and cognitive functions. Conversely, TBI can also stimulate neural stem/progenitor cell proliferation leading to increased numbers of neuroblasts migrating outside their restrictive neurogenic zone to areas of damage in support of tissue integrity. Unfortunately, the factors that regulate migration are poorly understood. Here, we examine whether ephrinB3 functions to restrict neuroblasts from migrating outside the subventricular zone (SVZ) and rostral migratory stream (RMS). We have previously shown that ephrinB3 is expressed in tissues surrounding these regions, including the overlying corpus callosum (CC), and is reduced after controlled cortical impact (CCI) injury. Our current study takes advantage of ephrinB3 knockout mice to examine the influences of ephrinB3 on neuroblast migration into CC and cortex tissues after CCI injury. Both injury and/or ephrinB3 deficiency led to increased neuroblast numbers and enhanced migration outside the SVZ/RMS zones. Application of soluble ephrinB3-Fc molecules reduced neuroblast migration into the CC after injury and limited neuroblast chain migration in cultured SVZ explants. Our findings suggest that ephrinB3 expression in tissues surrounding neurogenic regions functions to restrict neuroblast migration outside the RMS by limiting chain migration.

Oligodendrocyte birth and death following traumatic brain injury in adult mice.

Oligodendrocytes are responsible for producing and maintaining myelin throughout the CNS. One of the pathological features observed following traumatic brain injury (TBI) is the progressive demyelination and degeneration of axons within white matter tracts. While the effect of TBI on axonal health has been well documented, there is limited information regarding the response of oligodendrocytes within these areas. The aim of this study was to characterize the response of both mature oligodendrocytes and immature proliferative oligodendrocyte lineage cells across a 3 month timecourse following TBI. A computer-controlled cortical impact model was used to produce a focal lesion in the left motor cortex of adult mice. Immunohistochemical analyses were performed at 48 hours, 7 days, 2 weeks, 5 weeks and 3 months following injury to assess the prevalence of mature CC-1+ oligodendrocyte cell death, immature Olig2+ cell proliferation and longer term survival in the corpus callosum and external capsule. Decreased CC-1 immunoreactivity was observed in white matter adjacent to the site of injury from 2 days to 2 weeks post TBI, with ongoing mature oligodendrocyte apoptosis after this time. Conversely, proliferation of Olig2+ cells was observed as early as 48 hours post TBI and significant numbers of these cells and their progeny survived and remained in the external capsule within the injured hemisphere until at least 3 months post injury. These findings demonstrate that immature oligodendrocyte lineage cells respond to TBI by replacing oligodendrocytes lost due to damage and that this process occurs for months after injury.

Chemokines and inflammatory mediators interact to regulate adult murine neural precursor cell proliferation, survival and differentiation.

Adult neural precursor cells (NPCs) respond to injury or disease of the CNS by migrating to the site of damage or differentiating locally to replace lost cells. Factors that mediate this injury induced NPC response include chemokines and pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ), which we have shown previously promotes neuronal differentiation. RT-PCR was used to compare expression of chemokines and their receptors in normal adult mouse brain and in cultured NPCs in response to IFNγ and TNFα. Basal expression of many chemokines and their receptors was found in adult brain, predominantly in neurogenic regions, with OB≫SVZ>hippocampus and little or no expression in non-neurogenic regions, such as cortex. Treatment of SVZ-derived NPCs with IFNγ and TNFα (alone and in combination) resulted in significant upregulation of expression of specific chemokines, with CXCL1, CXCL9 and CCL2 most highly upregulated and CCL19 downregulated. Unlike IFNγ, chemokine treatment of NPCs in vitro had little or no effect on survival, proliferation or migration. Neuronal differentiation was promoted by CXCL9, CCL2 and CCL21, while astrocyte and total oligodendrocyte differentiation was not affected. However, IFNγ, CXCL1, CXCL9 and CCL2 promoted oligodendrocyte maturation. Therefore, not only do NPCs express chemokine receptors, they also produce several chemokines, particularly in response to inflammatory mediators. This suggests that autocrine or paracrine production of specific chemokines by NPCs in response to inflammatory mediators may regulate differentiation into mature neural cell types and may alter NPC responsiveness to CNS injury or disease.

Hyphal cell walls from the plant pathogen Rhynchosporium secalis contain (1,3/1,6)-beta-D-glucans, galacto- and rhamnomannans, (1,3;1,4)-beta-D-glucans and chitin.

A procedure has been developed for the isolation of cell walls from the hyphae of the causal agent for barley leaf scald, Rhynchosporium secalis (Oudem) J.J. Davis. Based primarily on monosaccharide linkage analysis, but also on the limited use of linkage-specific glucan hydrolases and solvent fractionation, the walls consist predominantly of (1,3/1,6)-beta-D-glucans, (1,3;1,4)-beta-D-glucans, galactomannans of (1,2;1,6)-Manp residues and (1,5)-galactofuranosyl [(1,5)-Galf] side chains, rhamnomannans of (1,6)-Manp residues and rhamnopyranosyl [(1,2)-Rhap] side chains, and chitin; the walls also contain approximately 23% (w/w) protein. Electron microscopy shows the presence of distinct inner and outer wall layers. Treatment of wall preparations with guanidine hydrochloride dissolves the outer layer and enables separate analysis of the inner and outer walls. The insoluble, inner wall layer is composed of (1,3/1,6)-beta-D-glucans, galacto- and rhamnomannans, (1,3;1,4)-beta-D-glucans and chitin, whereas the soluble outer wall material contains a high proportion of rhamnomannan, and smaller proportions of galactomannan, (1,3;1,4)-beta-D-glucan and (1,3/1,6)-beta-D-glucan with only trace levels of chitin. It was confirmed by immunochemical and enzymatic analysis that at least a portion of the (1,3;1,4)-beta-D-glucan component of the inner wall exists as a (1,3;1,4)-beta-D-glucan. The analyses not only provide information that is important for a complete understanding of the interactions between R. secalis and barley, but they also identify potential targets for the development of fungicides or resistant transgenic barley varieties.