Wisteria Floribunda Research Articles

P.1.010 - Brain-derived neurotrophic factor enhances glutamic acid decarboxylase and perineuronal net maturation in mouse primary cortical cultures

Schizophrenia is a debilitating mental illness which affects approximately 1% of the global population, and is characterised by positive (e.g. hallucinations) and negative (e.g. anhedonia) symptoms, combined with cognitive deficits. Evidence suggests diminished cortical inhibitory drive as a major contributing factor in schizophrenia pathophysiology. Reductions in glutamic acid decarboxylase (GAD65/67) and deficits in the extracellular matrix structures, perineuronal nets (PNNs), which enwrap parvalbumin-containing interneurons have been observed in patients [1,2]. Members of the c-Jun N-Terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) and their upstream kinase MKK7 have been implicated in neocortical development. Interestingly, MAP2K7 (encoding MKK7) has been implicated as a candidate risk gene for schizophrenia [3]. However, the impact of disrupted MAP2K7-JNK signalling on GABAergic interneuron development remains elusive. Reduced levels of brain-derived neurotrophic factor (BDNF) have also been found in patients [4]. Previous research highlights BDNF as a promoter of GABAergic maturation in hippocampal neurons [5]. Consequently, altered BDNF activity could result in abnormal cortical circuitry development and contribute to the cortical neuronal dysfunction underlying schizophrenia. Here we study the effects of BDNF on the development of primary cortical GABAergic interneurons and surrounding PNNs along with the possible involvement of JNK signalling. Mouse primary cortical neuronal cultures were treated at 7 days in vitro with either vehicle, BDNF (50 ng/ml), JNK inhibitor (SP600125, 5 μM) or BDNF+JNK inhibitor. Subsequently, immunofluorescent-cytochemistry (ICC) was performed. PNN development was assessed using Wisteria floribunda agglutinin (WFA) lectin which labels interneuron PNNs. Cultures were double-labelled with GAD65/67. Normalised intensity data (in relative optical density) were expressed as a percentage of vehicle and analysed via three-way ANOVA with Tukey post-hoc multiple comparisons with results considered significant if p Analysis of WFA staining intensity revealed significant main effects of BDNF (F1, 982 = 45.46, p Our data demonstrate that BDNF increased GAD65/67 levels and PNN density in developing cortical interneurons. Our data suggest this enhancing effect of BDNF on GAD65/67 and PNN maturation is dependent on JNK signalling. Given evidence of reduced BDNF levels in schizophrenia patients, we speculate that impairments in MAP2K7-JNK signalling may be linked with functional BDNF deficits in schizophrenia and that BDNF disruption could be involved in disorder aetiology and pathophysiology.

Amino acid substitutions in the hepatitis C virus core region predict hepatocarcinogenesis following eradication of HCV RNA by all-oral direct-acting antiviral regimens.

Impact of substitution of aa70 in the core region (Core aa70) in HCV genotype 1b (HCV-1b) on hepatocarcinogenesis following eradication of HCV RNA by direct-acting antiviral therapy is not clear. In a retrospective study, 533 patients with HCV-related chronic liver disease, with sustained virological response defined as negative HCV RNA at 12 weeks after cessation of direct-acting antiviral therapy, were examined to evaluate the relationship between Core aa70 substitution and hepatocarcinogenesis. Twelve patients developed hepatocellular carcinoma during the follow-up period. The cumulative hepatocarcinogenesis rates were 1.7% and 2.4% at the end of 1 and 2 years, respectively. Overall, multivariate analysis identified HCV subgroup (HCV-1b with Gln70(His70); P = 0.003) and age (>65 years; P = 0.049), as pretreatment predictors of hepatocarcinogenesis. In HCV-1b patients, multivariate analysis identified post-treatment Wisteria floribunda agglutinin positive Mac-2 binding protein (>1.8 COI; P = 0.042) and HCV subgroup (HCV-1b with Gln70(His70); P = 0.071), as predictors of hepatocarcinogenesis, including post-treatment parameter. In conclusion, Core aa70 substitution in HCV-1b at the start of direct-acting antiviral therapy is an important predictor of hepatocarcinogenesis following eradication of HCV RNA. This study emphasizes the importance of detection of Core aa70 substitution before initiating antiviral therapy.

(154) - Extracellular matrix mediated plasticity in neuropathic pain

In the nervous system, neurons and glial cells are embedded within an extracellular matrix (ECM), whose components not only provide structural support, but also regulate synapse formation and function, and modulate neuronal excitability. In the brain, ECM restricts synaptic and structural plasticity, and enzymatic digestion of ECM affects acquisition of memories, and promotes cognitive flexibility and extinction. In the pain pathway, matrix metalloproteinases (MMPs), the major ECM-digesting enzymes, are activated in neuropathic pain conditions to promote mechanical hypersensitivity; however, whether remodeling of spinal cord ECM contributes to pathological changes in dorsal horn nociceptive circuits and pain hypersensitivity remains unknown. We found that chronic inflammation and neuropathic pain lead to changes in the expression of ECM-related genes and to remodeling of the spinal ECM. To investigate the role of the ECM in restraining spinal plasticity, we injected chondroitinase ABC (chABC, an enzyme known to degrade ECM components) intrathecally before subjecting mice to Chronic Constriction Injury (CCI). Mice injected with chABC developed increased pain hypersensitivity as compared to that of vehicle-injected animals. Moreover, we found that Spared Nerve Injury (SNI) induces changes in spinal ECM chondroitin sulfate proteoglycans (CSPGs) such as Versican, Neurocan and Wisteria floribunda agglutinin (WFA)-binding proteins. In summary, our study demonstrates that peripheral nerve injury induces changes in the spinal ECM, suggesting a potential role of these modifications in the development of hypersensitivity.

Wisteria floribunda agglutinin-positive Mac-2 binding protein predicts the development of hepatocellular carcinoma in patients with non-alcoholic fatty liver disease.

As it is not practical to perform regular screening for hepatocellular carcinoma (HCC) in all patients with non-alcoholic fatty liver disease (NAFLD), there is a need to identify NAFLD patients who are at high risk for HCC. Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) has been shown to be a surrogate marker for predicting HCC as well as a liver fibrosis marker in patients with chronic hepatitis B and C. The aim of this study was to investigate whether WFA+ -M2BP predicts HCC development in NAFLD patients. Serum WFA+ -M2BP was retrospectively measured in 331 patients with histologically proven NAFLD, 51 of whom developed HCC. The association of WFA+ -M2BP and HCC development in NAFLD patients was investigated. The WFA+ -M2BP values were significantly greater in NAFLD patients with HCC than in those without HCC among patients with liver fibrosis ≥stage 3. Multivariate analysis identified WFA+ -M2BP as one of the predictive factors for HCC development (odds ratio, 1.57; 95% confidence interval, 1.083-2.265; P = 0.017). The optimal cut-off index of WFA+ -M2BP for predicting HCC was 1.255 with specificity of 78.4% and sensitivity of 70.4%. The area under the receiver operating characteristic curve value for the prediction of HCC development was 0.806. The cumulative incidence rate of HCC was significantly greater in patients with WFA+ -M2BP ≥ 1.255 (n = 61) than in those with WFA+ -M2BP < 1.255 (n = 137) among patients who were followed up for more than 2years after the diagnosis of NAFLD. Wisteria floribunda agglutinin-positive Mac-2 binding protein predicts HCC development and is a useful surrogate marker for identifying NAFLD patients who are at a high risk for HCC.

Expression of aggrecan components in perineuronal nets in the mouse cerebral cortex

Specific regions of the cerebral cortex are highly plastic in an organism’s lifetime. It is thought that perineuronal nets (PNNs) regulate plasticity, but labeling for Wisteria floribunda agglutinin (WFA), which is widely used to detect PNNs, is observed throughout the cortex. The aggrecan molecule—a PNN component—may regulate plasticity, and may also be involved in determining region-specific vulnerability to stress. To clarify cortical region-specific plasticity and vulnerability, we qualitatively analyzed aggrecan-positive and glycosylated aggrecan-positive PNNs in the mature mouse cerebral cortex. Our findings revealed the selective expression of both aggrecan-positive and glycosylated aggrecan-positive PNNs in the cortex. WFA-positive PNNs expressed aggrecan in a region-specific manner in the cortex. Furthermore, we observed variable distributions of PNNs containing WFA- and aggrecan-positive molecules. Together, our findings suggest that PNN components and their function differ depending on the cortical region, and that aggrecan molecules may be involved in determining region-specific plasticity and vulnerability in the cortex.

Structural Variation of Chondroitin Sulfate Chains Contributes to the Molecular Heterogeneity of Perineuronal Nets.

Aggrecan, a chondroitin sulfate (CS) proteoglycan, forms lattice-like extracellular matrix structures called perineuronal nets (PNNs). Neocortical PNNs primarily ensheath parvalbumin-expressing inhibitory neurons (parvalbumin, PV cells) late in brain development. Emerging evidence indicates that PNNs promote the maturation of PV cells by enhancing the incorporation of homeobox protein Otx2 and regulating experience-dependent neural plasticity. Wisteria floribunda agglutinin (WFA), an N-acetylgalactosamine-specific plant lectin, binds to the CS chains of aggrecan and has been widely used to visualize PNNs. Although PNNs show substantial molecular heterogeneity, the importance of this heterogeneity in neural plasticity remains unknown. Here, in addition to WFA lectin, we used the two monoclonal antibodies Cat315 and Cat316, both of which recognize the glycan structures of aggrecan, to investigate the molecular heterogeneity of PNNs. WFA detected the highest number of PNNs in all cortical layers, whereas Cat315 and Cat316 labeled only a subset of PNNs. WFA+, Cat315+, and Cat316+ PNNs showed different laminar distributions in the adult visual cortex. WFA, Cat315 and Cat316 detected distinct, but partially overlapping, populations of PNNs. Based on the reactivities of these probes, we categorized PNNs into four groups. We found that two subpopulation of PNNs, one with higher and one with lower WFA-staining are differentially labeled by Cat316 and Cat315, respectively. CS chains recognized by Cat316 were diminished in mice deficient in an enzyme involved in the initiation of CS-biosynthesis. Furthermore, WFA+ and Cat316+ aggrecan were spatially segregated and formed microdomains in a single PNN. Otx2 co-localized with Cat316+ but not with WFA+ aggrecan in PNNs. Our results suggest that the heterogeneity of PNNs around PV cells may affect the functional maturation of these cells.

Serum Wisteria floribunda agglutinin-positive Mac-2 binding protein more reliably distinguishes liver fibrosis stages in non-alcoholic fatty liver disease than serum Mac-2 binding protein.

Serum Mac-2 binding protein (M2BP) and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) are used to estimate the liver fibrosis stage in chronic liver diseases. However, few head-to-head studies have been carried out to compare the two biomarkers in non-alcoholic fatty liver disease (NAFLD). Serum M2BP and WFA+ -M2BP levels were compared against clinical characteristics and liver histological manifestations in the same samples collected from 213 biopsy-proven NAFLD patients. Median levels (range) of M2BP and WFA+ -M2BP were 1.58 (0.70-7.75) pg/mL and 0.85 (0.22-11.32) cut-off index (COI), respectively. Fibrosis stages 1, 2, 3, and 4 were determined in 136, 37, 17, and 23 patients, respectively. Median levels of both biomarkers increased stepwise with fibrosis progression. The M2BP and WFA+ -M2BP levels showed a significant positive correlation (r = 0.643, P = 2.91 × 10-26 ), but a marked discrepancy between both biomarkers was noted in five stage 4 and three stage 1 patients, who had high WFA+ -M2BP but relatively low M2BP levels. Most of these outliers had findings suggestive of more advanced fibrosis. For diagnosing any fibrosis severity, WFA+ -M2BP had greater area under the receiver operating characteristic curve (AUC) and predictive accuracy than M2BP. Among eight fibrosis markers/indices, WFA+ -M2BP yielded the second highest AUC (0.832) and the highest predictive accuracy (82.2%) to diagnose cirrhosis. In addition, WFA+ -M2BP showed the second highest predictive accuracy to diagnose severe fibrosis (78.4%) and significant fibrosis (76.1%). This head-to-head comparison suggests that WFA+ -M2BP is superior to M2BP for distinguishing liver fibrosis stages in NAFLD patients. A marked discrepancy between the two biomarkers may be indicative of advanced NAFLD (UMIN000023286).

WFA-labeled perineuronal nets in the macaque claustrum

ABSTRACTBackground: The claustrum (CLA) has been discussed as central to integrated conscious percepts, although recent evidence has emphasized a role in detecting sensory novelty or in amplifying correlated cortical inputs.Objective: We report that many neurons in the macaque CLA are ensheathed in perineuronal nets (PNNs), which contribute to synaptic stability and enhance neuronal excitability, among other properties.Design: We visualized PNNs by Wisteria floribunda agglutinin (WFA) immunohistochemistry, and quantified these in comparison these to parvalbumin+ (PV) subsets and total neurons.Results: PNNs ensheath about 11% of the total neurons. These are a range of large, medium, and small neurons, likely corresponding to PV+ and/or other inhibitory interneurons. The PNNs were themselves heterogeneous, consisting of lattice-like, weakly labeled, and diffuse subtypes, and showed some regional preference for the medial CLA.Conclusion: The abundant neuronal labeling by PNNs in the CLA suggests an important and nuanced role for inhibition, consistent with recent physiological studies of claustrocortical circuitry. For comparison, diversified inhibition in the reticular nucleus of the thalamus (a pan-inhibitory nucleus, with extensive cortical input) exerts a spectrum of control at different local and global spatiotemporal scales. Further investigation of PNN+ neurons in the macaque CLA offers a potentially important new approach to CLA function, relevant to the human brain both in normal and diseased conditions.

Consumption of a High-Fat Diet Alters Perineuronal Nets in the Prefrontal Cortex.

A key factor in the development of obesity is the overconsumption of fatty foods, which, in addition to facilitating weight gain, alters neuronal structures within brain reward circuitry. Our previous work demonstrates that sustained consumption of a high-fat diet (HFD) attenuates spine density in the prefrontal cortex (PFC). Whether HFD promotes structural adaptation among inhibitory cells of the PFC is presently unknown. One structure of interest is the perineuronal net (PNN), a specialized extracellular matrix surrounding, primarily, parvalbumin-containing GABAergic interneurons. PNNs contribute to synaptic stabilization, protect against oxidative stress, regulate the ionic microenvironment within cells, and modulate regional excitatory output. To examine diet-induced changes in PNNs, we maintained rats on one of three dietary conditions for 21 days: ad libitum chow, ad libitum 60% high fat (HF-AL), or limited-access calorically matched high fat (HF-CM), which produced no significant change in weight gain or adiposity with respect to chow controls. The PNN “number” and intensity were then quantified in the prelimbic (PL-PFC), infralimbic (IL-PFC), and ventral orbitofrontal cortex (OFC) using Wisteria floribunda agglutinin (WFA). Our results demonstrated that fat exposure, independent of weight gain, induced a robust decrease in the PNN intensity in the PL-PFC and OFC and a decrease in the PNN number in the OFC.

Large contribution of clonal reproduction to the distribution of deciduous liana species (Wisteria floribunda) in an old-growth cool temperate forest: evidence from genetic analysis.

Extensive clonal (vegetative) reproduction in lianas is a common and important life history strategy for regeneration and colonization success. However, few studies have evaluated the contribution of clonal reproduction to stand-level distribution of lianas in their natural habitat using genetic tools. The objectives of the present study were to investigate (1) the contribution of clonal reproduction to the distribution of Wisteria floribunda, (2) the size of clonal patches and (3) how the distribution patterns of W. floribunda clones are affected by micro-topography. The contribution of clonal reproduction to the distribution of the deciduous liana species W. floribunda was evaluated using genetic analysis across a 6-ha plot of an old-growth temperate forest in Japan and preference in landform between clonal ramets and non-clonal ramets was assessed. Of the 391 ramets sampled, clonal reproduction contributed to 71 and 62 % of the total abundance and basal area, respectively, or 57 and 31 % when the largest ramet within a genet was excluded. The large contribution of clonal reproduction to the density and basal area of W. floribunda was consistent with previous observational studies. The largest genet included a patch size of 0.47 ha and ranged over 180 m. Preferred landforms of clonal and non-clonal ramets were significantly different when evaluated by both abundance and basal area. Non-clonal ramets distributed more on lower part of the slope than other landforms in comparison with clonal ramets and trees, possibly reflecting the limitation of clonal growth by stolons. Using genetic analysis, the present study found evidence of a large contribution of clonal reproduction on the distribution of W. floribunda in its natural habitat. The results indicate that clonal reproduction plays an important role not only in the formation of populations but also in determining the distribution patterns of liana species.

Usefulness of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein in children with primary sclerosing cholangitis.

Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) is a novel serum marker of hepatic fibrosis in adults with chronic hepatitis C. However, it remains unclear whether serum WFA+ -M2BP levels are associated with the progression of liver histology in primary sclerosing cholangitis (PSC). Twenty-eight children and adolescents with pediatric-onset PSC (male:female patient ratio,20:8; median age at diagnosis, 9years) were enrolled in this study. The relation between serum WFA+ -M2BP levels and clinical characteristics was retrospectively evaluated. Moreover, receiver operating characteristic (ROC) analysis was used to determine whether serum WFA+ -M2BP levels could be a reliable marker to identify PSC patients with advanced liver histology. According to the Ludwig classification of liver histological stage, 28 patients were classified into the four stages. The WFA+ -M2BP level, aspartate aminotransferase (AST) to platelet ratio index (APRI), and hyaluronic acid correlated significantly with liver histological stage. Moreover, WFA+ -M2BP showed a significant positive correlation (P < 0.05) with autoimmune hepatitis overlap, AST, alanine aminotransferase (ALT), γ-glutamyltransferase, total bilirubin, immunoglobulin G, APRI, and hyaluronic acid. A ROC analysis was undertaken to distinguish the patients with advanced stage disease (stage 3-4) from those with early stage disease (stage 0-2). It showed that WFA+ -M2BP yielded the highest area under the ROC curve value (0.898) among four surrogate makers (APRI, 0.850; Fibrosis-4 index, 0.806; and AST/ALT ratio, 0.802). Moreover, WFA+ -M2BP yielded the highest sensitivity, specificity, positive predictive value, and negative predictive value among the four markers. Serum WFA+ -M2BP levels are useful to identify patients with advanced liver histology in pediatric PSC.

Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein can reflect systemic lupus erythematosus activity.

Serum Mac-2-binding protein (M2BP) is elevated in various chronic inflammatory diseases, and evidence suggests that glycosylation of M2BP induces discrete biological effects. However, the role of serum M2BP in systemic lupus erythematosus (SLE) is still unclear. Recently, a Wisteria floribunda agglutinin-positive-M2BP (WFA+-M2BP) immunoassay has shown promise in detecting highly glycosylated M2BP. In this study, by using WFA+-M2BP immunoassay, we measured serum M2BP in 203 SLE patients and evaluated its clinical significance. Eighty patients were classified as having active SLE and 123 patients as having inactive SLE. The median serum M2BP was higher in patients with active SLE than in those with inactive SLE (2.1 vs. 0.9, p < 0.001). In multivariate linear regression analysis, serum M2BP, anti-dsDNA, C3 and erythrocyte sedimentation rate (ESR) were associated with SLEDAI-2K. Serum M2BP also strongly correlated with laboratory variables related to SLEDAI-2K, ESR and C-reactive protein. Furthermore, multivariate logistic regression analysis demonstrated that serum M2BP was useful in predicting active SLE. Finally, following immunosuppressive treatment, elevated serum M2BP significantly decreased along with improvement in disease activity. These findings suggest that serum M2BP might contribute to the inflammatory process in SLE, and measuring serum M2BP might be a useful marker to assess SLE disease activity.

Age-dependent and region-specific alteration of parvalbumin neurons and perineuronal nets in the mouse cerebral cortex

Cognitive function declines with age. Such function depends on γ-oscillation in the frontal cortex. Pyramidal neurons, and the parvalbumin-expressing interneurons (PV neurons) that control them, are important for the generation of γ-oscillation. The mechanism by which cognitive function declines is unclear. Perineuronal nets (PNNs) mainly surround the soma and proximal dendrites and axon segments of PV neurons in the cerebral cortex. Previous evidence indicates that PNNs inhibit neural plasticity. If this is true, an increase in the number of neurons surrounded by PNNs or in the thickness or density of the PNNs around neurons could decrease plasticity in the cortex. To determine if an aging-related change in cortical PNNs occurs, we examined the influence of aging on PV neurons and whether Wisteria floribunda agglutinin-positive PNNs differ depending on the cortical area. The results showed that the number of PV neurons/mm2 did not change in many areas of the cortex as mice aged. In contrast, the number of neurons in the sensory cortex surrounded by PNNs increased as mice aged. Thus, with age, PNN density increases in some cortical areas but not in others. In addition, the expression level of PV protein in PV neurons decreased with aging in the whole cortex. We suggest that decreased expression of PV protein impairs fast spiking in PV neurons. We propose that PNNs surround more neurons as age increases. This aging-related increase in PNNs decreases plasticity in the cerebral cortex and reduces cognitive function. The first step in investigating this proposal would be to determine if PNN density increases with age.

Increased serum Wisteria floribunda agglutinin positive Mac-2 binding protein (Mac-2 binding protein glycosylation isomer) in chronic heart failure: a pilot study.

Serum Wisteria floribunda agglutinin positive Mac-2 binding protein (WFA+-M2BP) or Mac-2 Binding Protein Glycosylation Isomer (M2BPGi) is a novel biomarker currently applied for evaluating hepatic fibrosis. The aim of this study was to evaluate the utility of serum WFA+-M2BP level as a biomarker in chronic heart failure (HF) patients with abnormal liver function. Fifty chronic HF patients who underwent measurement of serum WFA+-M2BP were evaluated. The median value of serum WFA+-M2BP was 0.88 (interquartile range 0.48-1.29) cut-off index, and positive WFA+-M2BP (≥1.00 cut-off index) was observed in 22 (44%). Elevated WFA+-M2BP was associated with longer HF history, older age, female sex, valvular heart disease, decreased estimated glomerular filtration rate (eGFR), albumin, and cholinesterase. Stepwise multiple regression analysis showed that HF history, eGFR, and albumin were independent determinants of serum WFA+-M2BP values. Repeated measurements of serum WFA+-M2BP suggested association between the decrease of WFA+-M2BP and improvement of New York Heart Association (NYHA) functional class. Elevation of serum WFA+-M2BP showed a high prevalence in chronic HF patients with abnormal liver function with relation to HF history, decreased hepatic protein synthesis, and renal dysfunction. Our results suggest that serum WFA+-M2BP may be a novel biomarker of chronic HF.

Respiratory dysfunction following neonatal sustained hypoxia exposure during a critical window of brain stem extracellular matrix formation.

The extracellular matrix (ECM) modulates brain maturation and plays a major role in regulating neuronal plasticity during critical periods of development. We examined 1) whether there is a critical postnatal period of ECM expression in brain stem cardiorespiratory control regions and 2) whether the attenuated hypoxic ventilatory response (HVR) following neonatal sustained (5 days) hypoxia [SH (11% O2, 24 h/day)] exposure is associated with altered ECM formation. The nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus, hypoglossal motor nucleus, cuneate nucleus, and area postrema were immunofluorescently processed for aggrecan and Wisteria floribunda agglutinin (WFA), a key proteoglycan of the ECM and the perineuronal net. From postnatal day ( P) 5 ( P5), aggrecan and WFA expression increased postnatally in all regions. We observed an abrupt increase in aggrecan expression in the nTS, a region that integrates and receives afferent inputs from the carotid body, between P10 and P15 followed by a distinct and transient plateau between P15 and P20. WFA expression in the nTS exhibited an analogous transient plateau, but it occurred earlier (between P10 and P15). SH between P11 and P15 attenuated the HVR (assessed at P16) and increased aggrecan (but not WFA) expression in the nTS, dorsal motor nucleus of the vagus, and area postrema. An intracisternal microinjection of chondroitinase ABC, an enzyme that digests chondroitin sulfate proteoglycans, rescued the HVR and the increased aggrecan expression. These data indicate that important stages of ECM formation take place in key brain stem respiratory neural control regions and appear to be associated with a heightened vulnerability to hypoxia.

Mirror trends of plasticity and stability indicators in primate prefrontal cortex

Research on plasticity markers in the cerebral cortex has largely focused on their timing of expression and role in shaping circuits during critical and normal periods. By contrast, little attention has been focused on the spatial dimension of plasticity-stability across cortical areas. The rationale for this analysis is based on the systematic variation in cortical structure that parallels functional specialization and raises the possibility of varying levels of plasticity. Here, we investigated in adult rhesus monkeys the expression of markers related to synaptic plasticity or stability in prefrontal limbic and eulaminate areas that vary in laminar structure. Our findings revealed that limbic areas are impoverished in three markers of stability: intracortical myelin, the lectin Wisteria floribunda agglutinin, which labels perineuronal nets, and parvalbumin, which is expressed in a class of strong inhibitory neurons. By contrast, prefrontal limbic areas were enriched in the enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), known to enhance plasticity. Eulaminate areas have more elaborate laminar architecture than limbic areas and showed the opposite trend: they were enriched in markers of stability and had lower expression of the plasticity-related marker CaMKII. The expression of glial fibrillary acidic protein (GFAP), a marker of activated astrocytes, was also higher in limbic areas, suggesting that cellular stress correlates with the rate of circuit reshaping. Elevated markers of plasticity may endow limbic areas with flexibility necessary for learning and memory within an affective context, but may also render them vulnerable to abnormal structural changes, as seen in neurologic and psychiatric diseases.

Postnatal development of GABAergic interneurons and perineuronal nets in mouse temporal cortex subregions

In human neuropsychiatric disorders, there are functional and anatomical abnormalities of GABAergic interneurons in each temporal cortex subregion. Furthermore, accumulation of amyloid-β is observed in the temporal cortex in the early stages of Alzheimer’s disease. Each subregion of the temporal cortex has an important role in coordinating the input and output of the hippocampus. When subregions of the temporal cortex are impaired, memory and learning ability decrease. GABAergic interneurons control excitatory neurons, forming the cortico-cortical and cortico-hippocampal networks. However, in temporal cortex subregions, details of the distribution and developmental processes of GABAergic interneurons and perineuronal nets (PNNs) have not been elucidated. Here we examined the development of GABAergic interneurons and PNNs in mouse temporal cortex subregions. Results indicate that temporal cortex GABAergic interneurons have developmental stages different to those of the primary sensory cortex. In addition, the density of PNNs in the temporal cortex is lower than that in the sensory cortex. Furthermore, we found that the Wisteria floribunda agglutinin-reactive extracellular matrix molecule is present in the upper level of layer 1 of the temporal cortex. These results support the idea that mouse temporal cortex subregions develop differently from other cortical regions and have region-specific characteristics after maturation. The present study results suggested that the structure of the temporal cortex is significantly different from the sensory cortex and that temporal cortex may be highly vulnerable to neuropsychiatric and neurodegenerative disorders.

Use of Mac-2 binding protein as a biomarker for nonalcoholic fatty liver disease diagnosis.

In contrast to patients with viral hepatitis, patients with nonalcoholic fatty liver disease (NAFLD) can progress to hepatocellular carcinoma during the initial stages of liver fibrosis. Development and implementation of noninvasive methods for diagnosis and progression prediction are important for effective NAFLD surveillance. Mac‐2 binding protein (Mac‐2bp) is a useful nonalcoholic steatohepatitis (NASH) diagnosis biomarker and a powerful prediction biomarker for NAFLD fibrosis stage. Wisteria floribunda agglutinin (WFA)‐positive Mac‐2bp (WFA+‐M2BP) is a novel serum fibrosis biomarker for chronic hepatitis C that has clinical validity. Mac‐2bp and WFA+‐M2BP are also clinical NAFLD biomarker candidates. We examined the efficacy of Mac‐2bp and WFA+‐M2BP for NAFLD assessment using patients with biopsy‐proven NAFLD (n = 510; NAFLD cohort) and subjects who received a health check‐up (n = 2,122; check‐up cohort). In the NAFLD cohort, we set the fibrosis predicting cutoff values as 1.80 (F1), 2.21 (F2), and 2.24 μg/mL (F3). In the subjects with fatty liver from the check‐up cohort (n = 1,291), the serum Mac‐2bp levels were >1.80 μg/mL in 38.6% of the subjects (n = 498), and >2.24 μg/mL in 24.6% of the subjects (n = 318). The NAFLD cohort results indicated that Mac‐2bp and WFA+‐M2BP were equally useful for NASH diagnosis. During the early stages of fibrosis (F1, F2), the increase in Mac‐2bp was statistically significant but WFA+‐M2BP did not increase. Logistic regression analysis revealed that Mac‐2bp was an independent determinant for the prediction of advanced fibrosis stage (≥F2), even when adjusted for WFA+‐M2BP. Immunohistochemical staining of Mac‐2bp revealed that hepatocytes strongly expressed Mac‐2bp in patients with NAFLD. Conclusion: Our results indicated that hepatocyte‐derived Mac‐2bp would be a useful single biomarker for NASH diagnosis and fibrosis stage prediction in patients with NAFLD. (Hepatology Communications 2017;1:780–791)

Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep.

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke.

Hepatic stellate cells secreting WFA+ -M2BP: Its role in biological interactions with Kupffer cells.

Hepatic stellate cells (HSCs) play a central role in hepatic fibrosis and are regulated by Kupffer cells (KCs). Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+ -M2BP) was recently identified as a serum marker for hepatic fibrosis. Although WFA+ -M2BP was identified as a ligand of Mac-2, the function of WFA+ -M2BP in hepatic fibrosis remains unclear. Liver specimens were obtained from five patients with cirrhosis, five with chronic hepatitis, and five without hepatic fibrosis. WFA+ -M2BP kinetics were evaluated histologically and in subpopulations of liver cells such as HSCs, KCs, endothelial cells, biliary epithelial cells, and hepatocytes in in vitro culture. The function of WFA+ -M2BP in activated HSCs was evaluated using immunoblot analysis. Numbers of WFA+ -M2BP-positive cells in liver tissues increased with fibrosis stage. There were significant differences in WFA+ -M2BP levels between fibrosis stages F0 and F1-2 (P=0.012) and between fibrosis stages F1-2 and F3-4 (P<0.001). HSCs were the source of WFA+ -M2BP secretion in in vitro cultures of liver cells, as determined by sandwich immunoassay. Cells of the human HSC line LX-2 also secreted WFA+ -M2BP. Histologically, tissue sections showed that WFA+ -M2BP was located in Mac-2-expressing KCs. In vitro assays showed that exogenous WFA+ -M2BP stimulation enhanced Mac-2 expression in KCs and that HSCs co-cultured with KCs increased α-smooth muscle actin expression. Finally, Mac-2-depleted KCs with short interfering RNA had reduced α-smooth muscle actin expression following co-culturing with HSCs. WFA+ -M2BP from HSCs induces Mac-2 expression in KCs, which in turn activates HSCs to be fibrogenic.