Lihong Bu

GPR56 Regulates Pial Basement Membrane Integrity and Cortical Lamination

GPR56 is a member of the family of adhesion G-protein-coupled receptors that have a large extracellular region containing a GPS (G-protein proteolytic site) domain. Loss-of-function mutations in the GPR56 gene cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). BFPP is a radiological diagnosis and its histopathology remains unclear. This study demonstrates that loss of the mouse Gpr56 gene leads to neuronal ectopia in the cerebral cortex, a cobblestone-like cortical malformation. There are four crucial events in the development of cobblestone cortex, namely defective pial basement membrane (BM), abnormal anchorage of radial glial endfeet, mislocalized Cajal–Retzius cells, and neuronal overmigration. By detailed time course analysis, we reveal that the leading causal events are likely the breaches in the pial BM. We show further that GPR56 is present in abundance in radial glial endfeet. Furthermore, a putative ligand of GPR56 is localized in the marginal zone or overlying extracellular matrix. These observations provide compelling evidence that GPR56 functions in regulating pial BM integrity during cortical development.

Visual spatial memory is enhanced in female rats (but inhibited in males) by dietary soy phytoestrogens

BackgroundIn learning and memory tasks, requiring visual spatial memory (VSM), males exhibit superior performance to females (a difference attributed to the hormonal influence of estrogen). This study examined the influence of phytoestrogens (estrogen-like plant compounds) on VSM, utilizing radial arm-maze methods to examine varying aspects of memory. Additionally, brain phytoestrogen, calbindin (CALB), and cyclooxygenase-2 (COX-2) levels were determined.ResultsFemale rats receiving lifelong exposure to a high-phytoestrogen containing diet (Phyto-600) acquired the maze faster than females fed a phytoestrogen-free diet (Phyto-free); in males the opposite diet effect was identified. In a separate experiment, at 80 days-of-age, animals fed the Phyto-600 diet lifelong either remained on the Phyto-600 or were changed to the Phyto-free diet until 120 days-of-age. Following the diet change Phyto-600 females outperformed females switched to the Phyto-free diet, while in males the opposite diet effect was identified.Furthermore, males fed the Phyto-600 diet had significantly higher phytoestrogen concentrations in a number of brain regions (frontal cortex, amygdala & cerebellum); in frontal cortex, expression of CALB (a neuroprotective calcium-binding protein) decreased while COX-2 (an inducible inflammatory factor prevalent in Alzheimers disease) increased.ConclusionsResults suggest that dietary phytoestrogens significantly sex-reversed the normal sexually dimorphic expression of VSM. Specifically, in tasks requiring the use of reference, but not working, memory, VSM was enhanced in females fed the Phyto-600 diet, whereas, in males VSM was inhibited by the same diet. These findings suggest that dietary soy derived phytoestrogens can influence learning and memory and alter the expression of proteins involved in neural protection and inflammation in rats.

Neuroanatomic connectivity of the human ascending arousal system critical to consciousness and its disorders.

Abstract The ascending reticular activating system (ARAS) mediates arousal, an essential component of human consciousness. Lesions of the ARAS cause coma, the most severe disorder of consciousness. Because of current methodological limitations, including of postmortem tissue analysis, the neuroanatomic connectivity of the human ARAS is poorly understood. We applied the advanced imaging technique of high angular resolution diffusion imaging (HARDI) to elucidate the structural connectivity of the ARAS in 3 adult human brains, 2 of which were imaged postmortem. High angular resolution diffusion imaging tractography identified the ARAS connectivity previously described in animals and also revealed novel human pathways connecting the brainstem to the thalamus, the hypothalamus, and the basal forebrain. Each pathway contained different distributions of fiber tracts from known neurotransmitter-specific ARAS nuclei in the brainstem. The histologically guided tractography findings reported here provide initial evidence for human-specific pathways of the ARAS. The unique composition of neurotransmitter-specific fiber tracts within each ARAS pathway suggests structural specializations that subserve the different functional characteristics of human arousal. This ARAS connectivity analysis provides proof of principle that HARDI tractography may affect the study of human consciousness and its disorders, including in neuropathologic studies of patients dying in coma and the persistent vegetative state.

Equol an isoflavonoid: potential for improved prostate health, in vitro and in vivo evidence

BackgroundTo determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones.MethodsFirst, in vitro equol binding affinity for 5alpha-DHT was determined using 14C5alpha-DHT combined with cold 5alpha-DHT (3.0 nM in all samples). These steroids were incubated with increasing concentrations of equol (0-2,000 nM) and analyzed by Sephadex LH-20 column chromatography. 14C5alpha-DHT peak/profiles were determined by scintillation counting of column fractions. Using the 14C5alpha-DHT peak (0 nM equol) as a reference standard, a binding curve was generated by quantifying shifts in the 14C5alpha-DHT peaks as equol concentrations increased. Second, equols in vitro effects on LNCap cells were determined by culturing cells (48 hours) in the presence of increasing concentrations of dimethyl sulfoxide (DMSO) (vehicle-control), 5alpha-DHT, equol or 5alpha-DHT+equol. Following culture, prostate specific antigen (PSA) levels were quantified via ELISA. Finally, the in vivo effects of equol were tested in sixteen male Long-Evans rats fed a low isoflavone diet. From 190-215 days, animals received 0.1cc s.c. injections of either DMSO-control vehicle (n = 8) or 1.0 mg/kg (body weight) of equol (in DMSO) (n = 8). At 215 days, body and prostate weights were recorded, trunk blood was collected and serum assayed for luteinizing hormone (LH), 5alpha-DHT, testosterone and 17beta-estradiol levels.ResultsMaximum and half maximal equol binding to 5alpha-DHT occurred at approximately 100 nM and 4.8 nM respectively. LNCap cells cultured in the presence of 5alpha-DHT significantly increased PSA levels. However, in the presence of 5alpha-DHT+equol, equol blocked the significant increases in PSA levels from LNCap cells. In vivo equol treatment significantly decreased rat prostate weights and serum 5alpha-DHT levels but did not alter LH, testosterone, and estradiol levels.ConclusionsEquol administration appears to have potential beneficial effects for prostate health and other 5alpha-DHT mediated disorders. Equol administration: reduces PSA levels from LNCap cells under 5alpha-DHT stimulation, decreases rat prostate size, decreases serum 5alpha-DHT levels and androgen hormone action, while not altering other circulating sex steroids or LH levels.

Estrogens and phytoestrogens: brain plasticity of sexually dimorphic brain volumes

Sexually dimorphic brain volumes (sexually dimorphic nucleus of the preoptic area (SDN-POA) and anteroventral periventricular (AVPV) nucleus) are influenced by estrogens. Phytoestrogens, derived from plants (especially soy products), are molecules structurally and functionally similar to estradiol. The purpose of this study was to examine: the consumption of phytoestrogen (using a phytoestrogen-rich (Phyto-600) versus a phytoestrogen-free (Phyto-free)) diets from conception to adulthood (or changing the diets during adulthood) and characterizing (a) circulating plasma phytoestrogen levels, (b) testosterone levels in males, (c) sexually dimorphic brain volumes (i.e. the SDN-POA and AVPV) and (d) the presence of apoptotic cells in these brain structures in Long-Evans rats. Phyto-600 fed animals displayed total serum phytoestrogens levels 37-fold higher compared to Phyto-free values. Circulating testosterone levels were not significantly altered by the diets. Female SDN-POA volumes were not altered by the diets. Whereas, males fed a Phyto-free diet displayed decreased SDN-POA volumes compared to male Phyto-600 values. Females fed the Phyto-600 diet displayed larger AVPV volumes compared to males on the same diet or females on the Phyto-free diet. Males fed the Phyto-free diet had the largest AVPV values compared to Phyto-600 fed males. When the SDN-POA region was examined in lifelong Phyto-free fed males, apoptotic cells were present versus males fed the Phyto-600 diet and in the AVPV region the opposite results were obtained. In summary, consumption of dietary phytoestrogens (estrogen mimics) can alter hormone-sensitive hypothalamic brain volumes in rodents during adulthood.

Soy isoflavones modulate the expression of BAD and neuron-specific beta III tubulin in male rat brain.

Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. There is evidence that soy isoflavones influence neuronal apoptosis or proliferation in vitro and in vivo. However, little research has been done to investigate the effects of soy isoflavones on markers of neuronal apoptosis and survival in vivo. We examined brain BAD (a proapoptotic member of Bcl-2 protein family) and neuron-specific beta III tubulin (an early marker of neuronal differentiation/survival) levels in male rats fed either a standard chow rich of soy isoflavones (Phyto-600) or one free of soy isoflavones (Phyto-free) life-long (from conception until time tissue collected). Among five brain regions, the expression of BAD was highest in medial basal hypothalamus (MBH); the next highest in hippocampus; moderate in amygdala and frontal cortex; and lowest in cerebellum in Phyto-free fed animals. In animals on Phyto-600 diet, the levels of BAD were significantly decreased in frontal cortex and MBH; but significantly increased in the amygdala. The expression of beta III tubulin was highest in frontal cortex; moderate in amygdala, hippocampus and MBH; and lowest in cerebellum in the Phyto-free group. In rats fed with the Phyto-600 diet, levels of beta III tubulin were significantly increased in amygdala, frontal cortex, hippocampus and MBH compared to Phyto-free values. In summary, these findings provide evidence for the neuroprotective potential of soy isoflavones in the amygdala, frontal cortex, hippocampus and MBH. This implies that consumption of soy isoflavones may be beneficial on learning and memory, anxiety-related behaviors, and recovery from trauma.

Influences of dietary soy isoflavones on metabolism but not nociception and stress hormone responses in ovariectomized female rats

BackgroundIsoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. Few studies have examined the nociception and stress hormone responses after consumption of soy isoflavones.MethodsIn this study, ovariectomized (OVX) female Long-Evans rats were fed either an isoflavone-rich diet (Phyto-600) or an isoflavone-free diet (Phyto-free). We examined the effects of soy isoflavones on metabolism by measuring body weights, food/water intake, adipose tissue weights as well as serum leptin levels. Also, circulating isoflavone levels were quantified. During chemically induced estrous, nociceptive thresholds were recorded. Then, the animals were subjected to a stressor and stress hormone levels were quantified.ResultsBody weights were significantly lower in Phyto-600 fed rats compared to Phyto-free values within one week and during long-term consumption of soy isoflavones. Correspondingly, Phyto-600 fed animals displayed significantly less adipose deposition and lower serum leptin levels than Phyto-free values. However, rats on the Phyto-600 diet displayed greater food/water intake compared to Phyto-free levels. No changes in thermal pain threshold or stress hormone levels (ACTH and corticosterone) were observed after activation of the hypothalamic-pituitary-adrenal (HPA) stress axis.ConclusionIn summary, these data show that consumption of soy isoflavones 1) increases metabolism, demonstrated by significantly decreased body weights, adipose tissue deposition and leptin levels, but 2) does not alter nociception or stress hormone responses, as indexed by thermal pain threshold, serum corticosterone and ACTH levels in chemically-induced estrous OVX rats.

Stress (hypothalamic–pituitary–adrenal axis) and pain response in male rats exposed lifelong to high vs. low phytoestrogen diets

Estrogens exhibit complex but beneficial effects on brain structure, function and behavior. Soy-derived dietary phytoestrogens protect against hormone-dependent and age-related diseases, due to their estrogen-like hormonal actions. However, the effects of phytoestrogens on brain and behavior are relatively unknown. This study examined the influence of exposing male Long-Evans rats (lifelong) to either a phytoestrogen-rich (Phyto-600) or a phytoestrogen-free (Phyto-free) diet on body weights, behavioral pain thresholds, the hypothalamic-pituitary-adrenal (HPA) hormonal stress response, hippocampal glucocorticoid receptor and brain neural cell adhesion molecules (NCAM) and synaptophysin levels using standard behavioral and biochemical techniques. Body weights were significantly decreased in Phyto-600 fed animals compared to Phyto-free values. There were no significant changes in behavioral pain thresholds, circulating corticosterone concentrations (after acute immobilization stress) or NCAM and synaptophysin levels in various brain regions by the diet treatments. However, Phyto-600 fed males displayed significantly higher plasma adrenocorticotrophin (ACTH) (post-stress) and hippocampal glucocorticoid receptor levels vs. Phyto-free values. These data suggest that (1) body weights are significantly reduced by soy-derived phytoestrogens, (2) behavioral pain thresholds (via heat stimuli) are not influenced by dietary phytoestrogens, but (3) these estrogenic molecules in the hippocampus enhance glucocorticoid receptor abundance and alter the negative feedback of stress hormones towards a female-like pattern of higher ACTH release after activation of the HPA stress axis. This study is the first to show that lifelong consumption of dietary phytoestrogens alters the HPA stress response in male rats.

AVPV Neurons Containing Estrogen Receptor-Beta in Adult Male Rats Are Influenced by Soy Isoflavones

BackgroundIsoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. It is known that 17beta-estradiol induces apoptosis in anteroventral periventricular nucleus (AVPV) in rat brain. Also, there is evidence that consumption of soy isoflavones reduces the volume of AVPV in male rats. Therefore, in this study, we examined the influence of dietary soy isoflavones on apoptosis in AVPV of 150 day-old male rats fed either a soy isoflavone-free diet (Phyto-free) or a soy isoflavone-rich diet (Phyto-600).ResultsThe occurrence of apoptosis in AVPV was examined by TUNEL staining. The incidence of apoptosis was about 10 times higher in the Phyto-600 group (33.1 ± 1.7%) than in the Phyto-free group (3.6 ± 1.0%). Furthermore, these apoptotic cells were identified as neurons by dual immunofluorescent staining of GFAP and NeuN as markers of astrocytes and neurons, respectively. Then the dopaminergic neurons in AVPV were detected by immunohistochemistry staining of tyrosine hydroxylase (TH). No significant difference in the number of TH neurons was observed between the diet treatment groups. When estrogen receptor (ER) alpha and beta were examined by immunohistochemistry, we observed a 22% reduction of ERbeta-positive cell numbers in AVPV with consumption of soy isoflavones, whereas no significant change in ERalpha-positive cell numbers was detected. Furthermore, almost all the apoptotic cells were ERbeta-immunoreactive (ir), but not ERalpha-ir. Last, subcutaneous injections of equol (a major isoflavone metabolite) that accounts for approximately 70–90% of the total circulating plasma isoflavone levels did not alter the volume of AVPV in adult male rats.ConclusionIn summary, these findings provide direct evidence that consumption of soy isoflavones, but not the exposure to equol, influences the loss of ERbeta-containing neurons in male AVPV.

Effects of dietary phytoestrogens on core body temperature during the estrous cycle and pregnancy

Phytoestrogens have received increased investigative attention due to their potential protective effects in connection to age-related diseases and hormone-dependent cancers. Phytoestrogens appear to be an effective treatment during perimenopause where symptoms, such as hot flashes are reduced. However, little is known about the influence of phytoestrogens on core body temperature during various hormonal conditions. This study examined the effects of dietary phytoestrogens on core body temperature during estrous cycles or pregnancy by feeding Long-Evans rats either a diet rich in phytoestrogens (Phyto-600) versus a diet relatively low in phytoestrogens (Phyto-free). Independent of treatments, body temperature was highest at proestrus and declined during estrus and diestrus. Moreover, the consumption of the Phyto-600 diet moderately decreased body temperature during proestrus, estrus and diestrus versus Phyto-free-fed animals. During pregnancy, independent of treatments, core body temperature decreased as a function of increasing gestational length. Phyto-600-fed rats displayed significantly decreased body temperatures (by approximately 0.5 degrees C) from gestation days 6 to 19, compared to Phyto-free values. The results from this study indicate that consumption of dietary phytoestrogens alters the neuroendocrine mechanism of core body temperature regulation that may help explain, in part, the beneficial effects of phytoestrogens for hot flashes.