Strength exercise suppresses STZ-induced spatial memory impairment and modulates BDNF/ERK-CAMKII/CREB signalling pathway in the hippocampus of mice

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that has generated scientific interest because of its prevalence in the population. Studies indicate that physical exercise promotes neuroplasticity and improves cognitive function in animal models and in human beings. The aim of the present study was to investigate the effects of strength exercise on the hippocampal protein contents and memory performance in mice subjected to a model of sporadic AD induced by streptozotocin (STZ). Swiss mice received two injections of STZ (3 mg/kg, intracerebroventricular). After 21 days, they began physical training using a ladde. Mice performed this protocol for 4 weeks. After the last exercise training session, mice performed the Morris Water Maze test. The samples of hippocampus were excised and used to determine protein contents of brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase-Ca²⁺ (ERK), calmodulin-dependent protein kinase (CAMKII) and cAMP-response element-binding protein (CREB) signalling pathway. Strength exercise was effective against the decrease in the time spent and distance travelled in the target quadrant by STZ-injected mice. Strength exercise was also effective against the reduction of mature BDNF, tropomyosin receptor kinase B and neuronal nuclear antigen (NeuN) hippocampal protein levels in STZ mice. The decrease in the hippocampal ratio of pERK/ERK, pCAMKII/CAMKII and pCREB/CREB induced by STZ was reversed by strength exercise. Strength exercise decreased Bax/Bcl2 ratio in the hippocampus of STZ-injected mice. The present study demonstrates that strength exercise modulated the hippocampal BDNF/ERK-CAMKII/CREB signalling pathway and suppressed STZ-induced spatial memory impairment in mice.     

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn^−/0/Lum^−/− mouse model and single Lum^−/− or Bgn^−/0 mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn^−/0/Lum^−/− corneas demonstrated gross opacity that was not seen in the Bgn^−/0 or wild type corneas and greater than that in the Lum^−/− mice. The Bgn^−/0/Lum^−/− corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum^−/− and no opacity in Bgn^−/0 or wild type corneas. In the Bgn^−/0/Lum^−/− corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn^−/0 or wild type corneas and was restricted to the posterior stroma in Lum^−/− mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.     

Potential use of human adipose mesenchymal stromal cells for intervertebral disc regeneration: a preliminary study on biglycan-deficient murine model of chronic disc degeneration

Introduction

Biglycan is an important proteoglycan of the extracellular matrix of intervertebral disc (IVD), and its decrease with aging has been correlated with IVD degeneration. Biglycan deficient (Bgn^−/0) mice lack this protein and undergo spontaneous IVD degeneration with aging, thus representing a valuable in vivo model for preliminary studies on therapies for human progressive IVD degeneration. The purpose of the present study was to assess the possible beneficial effects of adipose-derived stromal cells (ADSCs) implants in the Bgn^−/0 mouse model.

Methods

To evaluate ADSC implant efficacy, Bgn^−/0 mice were intradiscally (L1-L2) injected with 8x10⁴ ADSCs at 16 months old, when mice exhibit severe and complete IVD degeneration, evident on both 7Tesla Magnetic Resonance Imaging (7TMRI) and histology. Placebo and ADSCs treated Bgn^−/0 mice were assessed by 7TMRI analysis up to 12 weeks post-transplantation. Mice were then sacrificed and implanted discs were analyzed by histology and immunohistochemistry for the presence of human cells and for the expression of biglycan and aggrecan in the IVD area.

Results

After in vivo treatment, 7TMRI revealed evident increase in signal intensity within the discs of mice that received ADSCs, while placebo treatment did not show any variation. Ultrastructural analyses demonstrated that human ADSC survival occurred in the injected discs up to 12 weeks after implant. These cells acquired a positive expression for biglycan, and this proteoglycan was specifically localized in human cells. Moreover, ADSC treatment resulted in a significant increase of aggrecan tissue levels.

Conclusion

Overall, this work demonstrates that ADSC implant into degenerated disc of Bgn^−/0 mice ameliorates disc damage, promotes new expression of biglycan and increased levels of aggrecan. This suggests a potential benefit of ADSC implant in the treatment of chronic degenerative disc disease and prompts further studies in this field.     

Lactobacillus rhamnosus GG and Bifidobacterium bifidum TMC3115 Can Affect Development of Hippocampal Neurons Cultured In Vitro in a Strain-Dependent Manner

This study examined whether Lactobacillus rhamnosus GG (LGG) and Bifidobacterium bifidum TMC3115 (TMC3115) could morphologically or physiologically influence hippocampal neuronal development in vitro. Hippocampal neurons cultured in vitro were exposed to live or heat-inactivated LGG or TMC3115 for either 6 or 24 h. Neuronal morphological changes and drebrin (DRB) and synaptophysin (SYP) protein levels were monitored using immunofluorescence. And the levels of DRB, SYP, and brain-derived neurotrophic factor (BDNF), and cAMP-response element binding protein (CREB) mRNA were detected using RT-PCR. The BDNF, CREB, and phosphorylated-CREB (P-CREB) protein levels were detected by extraction-enzyme-linked immunosorbent assay (ELISA) or Western blot assays. Heat-inactivated LGG and TMC3115 could enhance neuron viability, DRB and SYP protein levels, and BDNF mRNA level were significantly altered after exposure to the tested bacteria with 6 h or 24 h. There were no significant differences in neuronal morphology or DRB, SYP, or CREB mRNA levels among the groups following bacterial exposure. However, following exposure of live TMC3115 for 24 h, the neuronal BDNF and P-CREB protein levels were both significantly up-regulated as detected by western blot assays. These results demonstrated that LGG and TMC3115 could affect neuronal viability, along with hippocampal synaptic and functional development, in a strain-dependent manner, which may also be closely associated with the physiological and culture conditions of each strain. Up-regulated P-CREB may be one of the underlying mechanisms by which the bacteria, especially neurons following exposure of live TMC3115 for 24 h, are able to regulate neuronal BDNF protein production.

     

Genetic evidence for key roles of decorin and biglycan in dentin mineralization

Targeted disruption of the dentin sialophosphoprotein (DSPP) gene in the mice (Dspp^?/?) results in dentin mineralization defects with enlarged predentin phenotype similar to human dentinogenesis imperfecta type III. Using DSPP/biglycan (Dspp^?/?Bgn^?/0) and DSPP/decorin (Dspp^?/?Dcn^?/?) double knockout mice, here we determined that the enlarged predentin layer in Dspp^?/? teeth is rescued in the absence of decorin, but not in the absence of biglycan. However, Fourier transform infrared (FTIR) spectroscopy analysis reveals similar hypomineralization of dentin in both Dspp^?/?Bgn^?/0 and Dspp^?/?Dcn^?/? teeth. Atomic force microscopy (AFM) analysis of collagen fibrils in dentin shows subtle differences in the collagen fibril morphology in these genotypes. The reduction of enlarged predentin in Dspp^?/?Dcn^?/? mice suggests that the elevated level of decorin in Dspp^?/? predentin interferes with the mineralization process at the dentin mineralization front. On the other hand, the lack of DSPP and biglycan leads to the increased number of calcospherites in Dspp^?/?Bgn^?/0 predentin, suggesting that a failure in coalescence of calcospherites was augmented in Dspp^?/?Bgn^?/0 teeth as compared to Dspp^?/? teeth. These findings indicate that normal expression of small leucine rich proteoglycans, such as biglycan and decorin, plays an important role in the highly orchestrated process of dentin mineralization.     

Biglycan modulates angiogenesis and bone formation during fracture healing

Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (μCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.     

BDNF impairment is associated with age-related changes in the inner retina and exacerbates experimental glaucoma

Brain-derived neurotrophic factor (BDNF) stimulation of its high-affinity receptor TrkB results in activation of pro-survival cell-signalling pathways that can afford neuroprotection to the retina. Reduction in retrograde axonal transport of neurotrophic factors such as BDNF from the brain to the neuronal cell bodies in the retina has been suggested as a critical factor underlying progressive and selective degeneration of ganglion cell layer and optic nerve in glaucoma. We investigated the role of BDNF in preserving inner retinal homeostasis in normal and glaucoma states using BDNF^+/− mice and compared it with wild type controls. This study demonstrated that BDNF^+/− animals were more susceptible to functional, morphological and molecular degenerative changes in the inner retina caused by age as well as upon exposure to experimental glaucoma caused by increased intraocular pressure. Glaucoma induced a down regulation of BDNF/TrkB signalling and an increase in levels of neurotoxic amyloid β 1–42 in the optic nerve head which were exacerbated in BDNF^+/− mice. Similar results were obtained upon analysing the human optic nerve head tissues. Our data highlighted the role of BDNF in maintaining the inner retinal integrity under normal conditions and the detrimental effects of its insufficiency on the retina and optic nerve in glaucoma.     

Biglycan and chondroitin sulfate play pivotal roles in bone toughness via retaining bound water in bone mineral matrix

Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.     

Disturbances of systemic and hippocampal insulin sensitivity in macrophage migration inhibitory factor (MIF) knockout male mice lead to behavioral changes associated with decreased PSA-NCAM levels

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine well known for its role in inflammation enhancement. However, a growing body of evidence is emerging on its role in energy metabolism in insulin sensitive tissues such as hippocampus, a brain region implicated in cognition, learning and memory. We hypothesized that genetic deletion of MIF may result in the specific behavioral changes, which may be linked tо impairments in brain or systemic insulin sensitivity by possible changes of the hippocampal synaptic plasticity. To assess memory, exploratory behavior and anxiety, three behavioral tests were applied on Mif gene-deficient (MIF^−/−) and “wild type” C57BL/6J mice (WT). The parameters of systemic and hippocampal insulin sensitivity were also determined. The impact of MIF deficiency on hippocampal plasticity was evaluated by analyzing the level of synaptosomal polysialylated-neural cell adhesion molecule (PSA-NCAM) plasticity marker and mRNA levels of different neurotrophic factors.The results showed that MIF^−/− mice exhibit emphasized anxiety-like behaviors, as well as impaired recognition memory, which may be hippocampus-dependent. This behavioral phenotype was associated with impaired systemic insulin sensitivity and attenuated hippocampal insulin sensitivity, characterized by increased inhibitory Ser³⁰⁷ phosphorylation of insulin receptor substrate 1 (IRS1). Finally, MIF^−/− mice displayed a decreased hippocampal PSA-NCAM level and unchanged Bdnf, NT-3, NT-4 and Igf-1 mRNA levels.The results suggest that the lack of MIF leads to disturbances of systemic and hippocampal insulin sensitivity, which are possibly responsible for memory deficits and anxiety, most likely through decreased PSA-NCAM-mediated neuroplasticity rather than through neurotrophic factors.     

Brain-derived Neurotrophic Factor Regulates Energy Expenditure Through the Central Nervous System in Obese Diabetic Mice

It has been previously demonstrated that brain-derived neurotrophic factor (BDNF) regulates glucose metabolism and energy expenditure in rodent diabetic models such as C57BL/KsJ-lepr^db/lepr^db (db/db) mice. Central administration of BDNF has been found to reduce blood glucose in db/db mice, suggesting that BDNF acts through the central nervous system. In the present study we have expanded these investigations to explore the effect of central administration of BDNF on energy metabolism. Intracerebroventricular administration of BDNF lowered blood glucose and increased pancreatic insulin content of db/db mice compared with vehicle-treated pellet pair-fed db/db mice. While body temperatures of the pellet pair-fed db/db mice given vehicle were reduced because of restricted food supply in this pair-feeding condition, BDNF treatment remarkably alleviated the reduction of body temperature suggesting the enhancement of thermogenesis. BDNF enhanced norepinephrine turnover and increased uncoupling protein-1 mRNA expression in the interscapular brown adipose tissue. Our evidence indicates that BDNF activates the sympathetic nervous system via the central nervous system and regulates energy expenditure in obese diabetic animals.     

Cognition-enhancing and neuroprotective activities of the standardized extract of Betula platyphylla bark and its major diarylheptanoids

Diarylheptanoids have been the center of the intensive research efforts for Alzheimer's disease and other neurodegenerative diseases. The present study aimed to determine the effect of the standardized extract of B. platyphylla bark and its major diarylheptanoids in scopolamine-induced amnesic mice through cyclic AMP response element-binding protein (CREB) activation. Oral administration of the standardized extract of B. platyphylla bark (100 mg/kg body weight), aceroside VIII (1 mg/kg body weight) and platyphylloside (1 or 2 mg/kg body weight) significantly ameliorated scopolamine-induced amnesia in passive avoidance test. CREB phosphorylation and brain-derived neurotrophic factor (BDNF) expression in the cortex and hippocampus of the scopolamine-treated mice were markedly increased by the treatment of the standardized extract of B. platyphylla bark and platyphylloside. The standardized extract of B. platyphylla bark and its major diarylheptanoids also significantly protected HT22 cells against neurotoxicity induced by glutamate insult. The standardized extract of B. platyphylla bark and platyphylloside may ameliorate memory deficits by activating the CREB–BDNF pathway and prevent a neurodegeneration by inhibiting neuronal cell death.     

Norepinephrine induces BDNF and activates the PI-3K and MAPK cascades in embryonic hippocampal neurons

Both antidepressant treatment and physical exercise have been shown to increase circulating levels of norepinephine (NE) and hippocampal brain-derived neurotrophic factor (BDNF). Increases in BDNF have been shown to be associated with enhanced dendritic arborization and neuronal survival, which forms the theoretical basis of the Neurotrophin Hypothesis of antidepressant action. Using isolated embryonic hippocampal neurons and immunoblotting, we show that application of NE increases BDNF and phosphorylated Trk, and that these increases can be prevented by ERK and PI-3K inhibitors. In addition, NE-induced increases in phospho-ERK2 and PI-3K were each suppressed by a PI-3K and MAPK inhibitor, respectively. Furthermore, phosphorylation of cAMP-response element binding (CREB) protein was also increased by NE and brought down to baseline levels by MAPK and PI-3K inhibitors. And finally, because both the MAPK and PI-3K inhibitors suppress phosphorylation of both TrkB (upstream) and CREB (downstream), these results indicate that NE-induced BDNF expression follows a cyclic pathway, reminiscent of a positive feedback loop. The results of this study provide an in vitro model of the intracellular signaling mechanisms activated by NE, via ligand-G-protein-coupled receptor (GPCR)-to-BDNF-RTK transactivation, that is putatively thought to occur in vivo as a result of excitatory neural activity.