Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum.

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aß) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes ß-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aß. Finally, we show the ammonia-induced production of Aß in astrocytic endoplasmic reticulum was specific to Aß42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aß42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.

Estimation of Behavior Change Stage from Walking Information and Improvement of Walking Volume by Message Intervention.

Lifestyle-related diseases are a major problem all over the world although exercising can prevent them. Therefore, it is necessary to encourage users to exercise regularly and to support their exercises. The purpose of this study is to investigate whether the estimation of behavior change stages can be predicted from the gait information obtained from wearable devices, and whether message interventions created based on the behavior change stages are effective in improving the amount of walking. As for the estimation of the behavior change stages, we investigated whether the behavior change stages could be correctly estimated compared with the ones obtained from the questionnaire. As for the effect of the message, we compared the period of no intervention with that of intervention to examine whether there was any change in the amount of walking. As a result of the experiment, we could not properly estimate the behavior change stage of users, but we found that the message intervention improved the amount of walking for many subjects. This suggests that further research is needed to estimate the stage of behavior change. However, message intervention is confirmed as an effective means to improve walking volume.

Contribution of synovial lining cells to synovial vascularization of the rat temporomandibular joint.

The lining layer of the synovial membrane in the temporomandibular joint (TMJ) contains two types of lining cells: macrophage-like type A and fibroblast-like type B cells. The type B cells are particularly heterogeneous in their morphology and immunoreactivity, so that details of their functions remain unclear. Some of the type B cells exhibit certain resemblances in their ultrastructure to those of an activated capillary pericyte at the initial stage of the angiogenesis. The articular surface, composed of cartilage and the disc in the TMJ, has few vasculatures, whereas the synovial lining layer is richly equipped with blood capillaries to produce the constituent of synovial fluid. The present study investigated at both the light and electron microscopic levels the immunocytochemical characteristics of the synovial lining cells in the adult rat TMJ, focusing on their contribution to the synovial vascularization. It also employed an intravascular perfusion with Lycopersicon esculentum (tomato) lectin to identify functional vessels in vivo. Results showed that several type B cells expressed desmin, a muscle-specific intermediate filament which is known as the earliest protein to appear during myogenesis as well as being a marker for the immature capillary pericyte. These desmin-positive type B cells showed immunoreactions for vimentin and pericyte markers (neuron-glial 2; NG2 and PDGFRß) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (αSMA and caldesmon). Immunoreactivity for RECA-1, an endothelial marker, was observed in the macrophage-like type A cells. The arterioles and venules inside the synovial folds extended numerous capillaries with RECA-1-positive endothelial cells and desmin-positive pericytes to distribute densely in the lining layer. The distal portion of these capillaries showing RECA-1-immunoreactivity lacked lectin-staining, indicating a loss of blood-circulation due to sprouting or termination in the lining layer. The desmin-positive type B and RECA-1-positive type A cells attached to this portion of the capillaries. Some capillaries in the lining layer also expressed ninein, a marker for sprouting endothelial cells, called tip cells. Since an activated pericyte, macrophage and tip cell are known to act together at the forefront of the vessel sprout during angiogenesis, the desmin-positive type B cell and RECA-1-positive type A cell might serve as these angiogenic cells in the synovial lining layer. Tomato lectin perfusion following decalcification would be a highly useful tool for research on the vasculature of the mineralized tissue. Use of this technique combined with immunohistochemistry should permit future extensive investigations on the presence of the physiological angiogenesis and on the function of the lining cells in the synovial membrane.

Immunoexpression of aquaporin-1 in the rat periodontal ligament during experimental tooth movement.

This study examined the immunoexpression pattern of aquaporin-1 (AQP1), first identified as a water channel protein, in the periodontal ligament of rat molars during experimental tooth movement to clarify its role in periodontal responses in an overloaded model by the insertion of a piece of elastic band. In the control group without any treatment, the cementoblasts and osteogenic cells as well as the vascular endothelial cells showed AQP1 immunoreaction. In the experimental group, hyalinized tissue and intensely AQP1 positive amorphous structures which were identified as degenerated endothelial cells by immunoelectron microscopy, occurred at the compression side on Days 1 and 3. AQP1 immunoreaction came to be stronger in the intact endothelial cells around the hyalinized tissue. The hyalinized tissue had almost disappeared by Day 5 when many macrophages reactive to acid phosphatase activity appeared. The periodontal width on Day 7 became almost the same as that in the control group. These findings indicate that the hyalinized tissue and damaged AQP1 positive endothelial cells are phagocytized by macrophages which have temporally migrated, and suggest that the surviving endothelial cells with intense AQP1 reaction are involved in periodontal regeneration by capillary sprouting.

Detection of acid-sensing ion channel 3 (ASIC3) in periodontal Ruffini endings of mouse incisors.

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion - which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings.

GDNF expression in terminal schwann cells associated with the periodontal ruffini endings of the rat incisors during nerve regeneration.

The terminal Schwann cells (TSCs) which play crucial roles in regeneration of the periodontal Ruffini endings (RE) exhibit immunoreaction for glial cell line-derived neurotrophic factor (GDNF). However, no information is available regarding the role of GDNF in the periodontal RE during nerve regeneration. This study was undertaken to examine the changes in GDNF expression in the rat periodontal RE following transection of the inferior alveolar nerve (IAN) using immunohistochemistry for GDNF and S-100 protein, a marker for the TSCs. We additionally investigated the changes in expression of GDNF in the trigeminal ganglion (TG) at protein and mRNA levels. A transection to IAN induced a disappearance of the TSCs from the alveolus-related part (ARP), followed by a migration of spindle-shaped cells with S-100 but without GDNF immunoreactions into the tooth-related part (TRP) by postoperative (PO) week 2. At PO week 2, GDNF immunoreacted cellular elements increased in number in the ARP although the spindle-shaped cells without GDNF reaction remained in the TRP. After PO week 4, many GDNF-positive TSCs appeared in the ARP though the spindle-shaped cells vanished from the TRP. A real time RT-PCR analysis demonstrated the highest elevation of GDNF mRNA in the TG at PO week 2. These findings suggested the involvement of this molecule in the maturation and maintenance of the periodontal RE during regeneration. Taken together with our previous and current studies, it appears that the regeneration of the periodontal RE is controlled by multiple neurotrophins in a stage-specific manner.

Involvement of neurotrophin-4/5 in regeneration of the periodontal Ruffini endings at the early stage.

Little is known about the role of neurotrophin-4/5 (NT-4/5) in the regeneration of mechanoreceptors. Therefore, the present study examined the regeneration process of Ruffini endings in the periodontal ligament in nt-4/5-deficient and wildtype mice following transection of the inferior alveolar nerve by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by computer-assisted quantitative image analysis. Furthermore, rescue experiments by a continuous administration of recombinant NT-4/5 were performed and analyzed quantitatively. At postoperative day 3 (PO 3d), almost all PGP 9.5-positive neural elements had disappeared; they began to appear in both types of animals at PO 7d. At PO 10d, almost all nerve fibers showed a beaded appearance, with fewer ramifications in both types of mice. Although the regeneration proceeded in the wildtype, a major population of the periodontal Ruffini endings continued to display smooth outlines at PO 28d in the nt-4/5 homozygous mice. The reduction ratio of neural density reached a maximum at PO 3d, decreased at PO 10d, and later showed a plateau. In a rescue experiment, an administration of NT-4/5 showed an acceleration of nerve regeneration in the homozygous mice. These findings indicate that the nt-4/5-depletion causes a delay in the regeneration of the periodontal Ruffini endings, but the delay is shortened by an exogenous administration of NT-4/5. Combined with our previous findings of bdnf-deficient mice (Harada et al. [2003] Arch Histol Cytol 66:183-194), these morphological and numerical data suggest that multiple neurotrophins such as NT-4/5 and brain-derived neurotrophic factor (BDNF) play roles in their regeneration in a stage-specific manner.

Neurotrophin-4/5-depletion induces a delay in maturation of the periodontal Ruffini endings in mice.

Neurotrophin-4/5 (NT-4/5) - a member of the neurotrophic factors - is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wild-type and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75- NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT- 4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.

Involvement of brain-derived neurotrophic factor (BDNF) in the development of periodontal Ruffini endings.

The periodontal Ruffini ending has been reported to show immunoreactivity for tyrosine kinase B (trkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the periodontal ligament of the rat incisor. Furthermore, adult heterozygous BDNF-mutant mice showed malformation and reduction of the periodontal Ruffini endings. To investigate further roles of BDNF in these structures, the development, distribution, and terminal morphology of Ruffini endings were examined in the incisor periodontal ligament of heterozygous and homozygous BDNF mutant mice, as well as in the wild-type littermate by immunohistochemistry for protein gene product (PGP) 9.5, a general neuronal marker. A similar distribution and terminal formation of PGP 9.5-immunoreactive nerve fibers was recognized in the periodontal ligament of all phenotypes at postnatal week (PW) 1. At this stage, the nerve fibers had a beaded appearance, but did not form the periodontal Ruffini endings. At PW2, the heterozygous and wild-type mice started to show ramified nerve fibers resembling the mature shape of periodontal Ruffini endings. At PW3, the Ruffini endings occurred in the periodontal ligament of the wild-type and heterozygous mice. While the Ruffini endings of the wild-type mice appeared either ruffled or smooth, as reported previously, most of these structures showed a smooth outline in the heterozygous mice. The homozygous mice lacked the typical Ruffini endings at PW3. In the quantitative analysis, homozygous mice had the smallest percentages of PGP 9.5-immunoreactive areas at the same postnatal periods, but there were no significant differences between wild-type and heterozygous mice during PW1-3. These findings suggest a possible involvement of BDNF during the postnatal development and, in particular, the maturation of periodontal Ruffini endings. Furthermore, other neurotrophins may play a role in the development and/or early maturation of the periodontal nerve fibers, as indicated by the presence of nerve fibers in the BDNF-homozygous mice.

The involvement of brain-derived neurotrophic factor (BDNF) in the regeneration of periodontal Ruffini endings following transection of the inferior alveolar nerve.

The present study employed immunohistochemistry for protein gene product 9.5 (PGP 9.5) to examine the regeneration process of Ruffini endings, the primary mechanoreceptor in the periodontal ligament, in heterozygous mice with targeted disruption of the brain-derived neurotrophic factor (BDNF) gene and their littermates, following transection of the inferior alveolar nerve. When immunostained for PGP 9.5, periodontal Ruffini endings appeared densely distributed in the periodontal ligament of the heterozygous mice, but the density of the positively stained nerve fibers in the ligament was 20% lower than that in the control littermates. At 3 days after surgery, the PGP 9.5-positive neural elements had disappeared; they began to appear in the periodontal ligament of both animals at 7 days. However, the recovery pattern of the PGP 9.5-positive nerves differed between heterozygous and wild type mice, typical periodontal Ruffini endings morphologically identical to those in the control group appeared in the wild-type mice at 7 days, whereas such Ruffini endings were detectable in the heterozygous mice at 28 days, though much smaller in number. On day 28, when PGP 9.5-positive nerves were largely regenerated in wild type mice, their distribution was much less dense in the ligament of the heterozygous mice than in the non-treated heterozygous mice. The density of PGP 9.5-positive nerve fibers was significantly lower in the heterozygous mice than in wild type mice at any stage examined. These data showing that a reduced expression of BDNF causes delayed regeneration of the periodontal Ruffini endings suggest the involvement of BDNF in the regeneration process of these mechanoreceptors.

Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve.

The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25-immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively.

The periodontal Ruffini endings in brain derived neurotrophic factor (BDNF) deficient mice.

Innervation and terminal morphology in the lingual periodontal ligament of the incisor were investigated in brain derived neurotrophic factor (BDNF) heterozygous mice and littermate wild-type mice (aged two months) using immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker. In addition, computer-assisted quantitative analysis was performed for a comparison of neuronal density in the periodontal ligament between heterozygous and wild-type mice. In wild-type mice, the periodontal ligament was found to be richly innervated by the mechanoreceptive Ruffini endings and nociceptive free nerve endings in the alveolus-related part of the periodontal ligament. The periodontal Ruffini endings in the wild-type mice incisor ligament were classified into two types: type I with ruffled outlines, and type II with a smooth outline. BDNF heterozygous mice showed malformations of the type I Ruffini endings which included fewer nerve fibers and fewer ramifications than those in wild-type mice as well as smooth outlines of the axon terminals. Quantitative analysis under a confocal microscope showed a roughly 18% reduction in neuronal density in the periodontal ligament of the heterozygous mice. These findings suggest that the development and maturation of the periodontal Ruffini endings require BDNF.