A rescue factor for Alzheimer's diseases: discovery, activity, structure, and mechanism.

Alzheimer's disease (AD) is a complex disease, involving multiple factors such as the production of aggregation-prone amyloid beta (Abeta) peptides, the formation of fibrillarly tangles of microtubule-associating proteins, Tau, and the polymorphism of cholesterol binding protein, APOE4. While understanding the mechanism of AD and the involvement of key players should lead to rational drug discovery against this disease, a traditional screening approach should also work for identifying drugs using AD models. We have used a cellular AD model, in which a cell death was induced by AD-causing neurotoxicities, and then screened the genes, which rescued the cells from the cell death. This resulted in isolation of a gene encoding a novel 24-amino acid long peptide, termed Humanin (HN), which protected neuronal cells at approximately microM level. Surprisingly, these gene products and the synthetic peptides not only protected neurons from cell death induced by Abeta-related neurotoxicities, but also Abeta-unrelated neurotoxicities. While a broad range of activities of HN against AD-related insults is discovered, the detailed mechanism of its action is still obscure. Structure analysis of HN showed that it is largely disordered and flexible at low peptide concentrations and heavily aggregates at high concentrations. Interestingly, one of the HN analogs, which is 10000-times more active than the parent HN molecule (i.e. active below nM range), was found to be monomeric. Based on findings of structural analyses, we propose here that membrane environment may enable HN to achieve high affinity for target protein(s) with multiple-transmembrane domains, such as G-protein coupled receptors.

Altered expression of microRNA during fracture healing in diabetic rats.

OBJECTIVES: Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. METHODS: Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. RESULTS: Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. CONCLUSIONS: Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM.Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139-147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1.

Clinicopathologic Impact of Early Medullary Ray Injury in Patients Following Kidney Transplantation.

BACKGROUND: Previously, we explored the histopathologic characteristics of medullary ray injury (MRI) inducing interstitial fibrosis and tubular atrophy (IF/TA) to determine its etiologies, which include calcineurin inhibitor (CNI) toxicity and urologic complications. However, we did not examine the effects of these etiologies on long-term kidney allograft prognosis, because biopsy timing differed among cases. AIM: We examined the influence of early MRI on kidney allograft prognosis using protocol biopsies taken within a 3-month time frame. METHODS: We defined early MRI as tubular degeneration with interstitial edema or mild fibrosis localized to the medullary ray. We divided 53 protocol biopsies into 2 groups, with and without early MRI. Early MRI+ cases with isometric vacuolization were classified as CNI toxicity; those with Tamm-Horsfall protein in the interstitium and a thyroidlike appearance were classified as urinary tract system abnormalities; remaining cases were classified as "others." We compared changes in serum levels of creatinine (sCr) over 3 years and fibrosis extent at 1 year. RESULTS: The sCr levels were significantly higher in the MRI+ group than the MRI- group at 3 years (P = .024). Examining the 3 MRI+ subgroups, only the MRI+ urinary tract system abnormalities group had significantly high sCr levels compared to the MRI- group (P = .019). The MRI+ group showed significant signs of IF/TA at 1 year. CONCLUSIONS: Early MRI after kidney transplantation was significantly more likely to develop IF/TA at 1 year and had higher sCr levels at 3 years. In such cases, intervention might preserve graft function over the long term.

Osteogenic potential of cells in vitro derived from haemarthrosis of the knee induced by injury to the anterior cruciate ligament.

We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with beta-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH)(2)D(3), a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and mRNA levels in osteoblast-related genes were enhanced. Our results suggest that the haemarthrosis induced by injury to the anterior cruciate ligament contains osteoprogenitor cells and is a potential alternative source for cell-based treatment in such injury.

Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases.

The epsilon4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 microg/ml ApoE4, but not by 30 microg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, alpha2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Galpha(i) restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G(i) class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.

Insulin-like growth factor I (IGF-I) protects cells from apoptosis by Alzheimer's V642I mutant amyloid precursor protein through IGF-I receptor in an IGF-binding protein-sensitive manner.

It has been found that insulin-like growth factor I (IGF-I) exerts cytoprotection against Abeta amyloid-induced neuronal cell death. Deposits of Abeta amyloid are one of the pathological hallmarks of Alzheimer's disease (AD). Here, we examined whether IGF-I exerts protective activity against cell death induced by a familial AD (FAD)-linked mutant of amyloid precursor protein (APP), and we found that IGF-I protected cells from toxicity of FAD-associated V642I mutant of APP in multiple cell systems. IGFBP-3 blocked this action of IGF-I, but not of des(1-3)IGF-I, which was as active as IGF-I in the presence of IGFBP-3. The data also demonstrated that the IGF-I receptor (IGF-IR) mediates the protective activity of IGF-I. The antagonizing function of the IGF-I/IGF-IR system against V642I-APP, which is further antagonized by IGFBP-3, provides a molecular clue to the understanding of AD pathophysiology and to the establishment of potential therapy for AD.

Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults.

A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer's disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by Abeta1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other Abeta peptides (Abeta1-42 and Abeta25-35). The action specificity was further indicated by the finding that HN could not suppress neurotoxicity by glutamate or prion fragment. Against the AD-relevant insults, essential roles of Cys(8) and Ser(14) were commonly indicated, and the domain from Pro(3) to Pro(19) was responsible for the rescue action of HN, in which seven residues turned out to be essential. We also compared the neuroprotective action of S14G HN (HNG) with that of activity-dependent neurotrophic factor, IGF-I, or basic FGF for the antagonism against various AD-relevant insults (V642I-APP, NL-APP, M146L-PS1, N141I-PS2, and Abeta1-43). Although all of these factors could abolish neurotoxicity by Abeta1-43, only HNG could abolish cytotoxicities by all of them. HN and HN derivative peptides may provide a new insight into the study of AD pathophysiology and allow new avenues for the development of therapeutic interventions for various forms of AD.

c-Jun N-terminal kinase (JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK.

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

Profiling microRNA expression in fracture nonunions: Potential role of microRNAs in nonunion formation studied in a rat model.

MicroRNAs (miRNAs ) are small non-coding RNAs that regulate gene expression. We hypothesised that the functions of certain miRNAs and changes to their patterns of expression may be crucial in the pathogenesis of nonunion. Healing fractures and atrophic nonunions produced by periosteal cauterisation were created in the femora of 94 rats, with 1:1 group allocation. At post-fracture days three, seven, ten, 14, 21 and 28, miRNAs were extracted from the newly generated tissue at the fracture site. Microarray and real-time polymerase chain reaction (PCR) analyses of day 14 samples revealed that five miRNAs, miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p, were highly upregulated in nonunion. Real-time PCR analysis further revealed that, in nonunion, the expression levels of all five of these miRNAs peaked on day 14 and declined thereafter. Our results suggest that miR-31a-3p, miR-31a-5p, miR-146a-5p, miR-146b-5p and miR-223-3p may play an important role in the development of nonunion. These findings add to the understanding of the molecular mechanism for nonunion formation and may lead to the development of novel therapeutic strategies for its treatment.

Complete traumatic backout of the blade of proximal femoral nail antirotation: a case report.

This article presents a rare case of traumatic complete expulsion of the helical blade after successful treatment of an intertrochanteric fracture with proximal femoral nail antirotation (PFNA). A 94-year-old woman sustained an intertrochanteric fracture of the left femur. Fracture fixation was performed by using PFNA-II. At six months FU, the patient presented with pain at the proximal lateral left thigh after she had fallen. A protrusion was noted. Radiographs showed a complete expulsion of the helical blade with a healed intertrochanteric fracture. The PFNA-II was removed and a cemented bipolar hemiarthroplasty was performed. At 5 months after surgery, the patient was able to walk with a walker without pain. Traumatic complete expulsion of the blade should be considered as a possible complication of PFNA/PFNA-II.

The osteogenic activity of human mandibular fracture haematoma-derived cells is stimulated by low-intensity pulsed ultrasound in vitro.

Low intensity pulsed ultrasound (LIPUS) stimulation is a clinically established treatment method used to accelerate long bone fracture healing; however, this method is currently not applied to mandibular fractures. In this study, we investigated the effects of LIPUS on human mandibular fracture haematoma-derived cells (MHCs) in order to explore the possibility of applying LIPUS treatment to mandibular fractures. MHCs were isolated from five patients. The cells were divided into two groups: (1) LIPUS (+) group: MHCs cultured in osteogenic medium with LIPUS treatment; and (2) LIPUS (-) group: MHCs cultured in osteogenic medium without LIPUS treatment. The osteogenic differentiation potential and proliferation of the MHCs were compared between the two groups. The waveform used was equal to the wave conditions of a clinical fracture healing system. The gene expression levels of ALP, OC, Runx2, OSX, OPN, and PTH-R1 and mineralization were increased in the LIPUS (+) group compared to the LIPUS (-) group. There were no significant differences in cell proliferation between the two groups. These findings demonstrate the significant effects of LIPUS on the osteogenic differentiation of MHCs. This study provides significant evidence for the potential usefulness of the clinical application of LIPUS to accelerate mandibular fracture healing.

Advances in characterization of neuroprotective peptide, humanin.

Humanin (HN), a short amino acid peptide, protects neurons as well as other cells from amyloid ß-induced toxicities and other stresses. A number of HN binding proteins have been identified and their involvements in HN-mediated neuroprotection have been suggested in some cases. However, the way HN binds to the target molecules has never been clarified. Here we will review the structures of HN and HN analogs in solution as a function of solvent conditions and attempt to relate their structural characteristics to the functional properties.

Efficient cell-seeding into scaffolds improves bone formation.

Bone marrow stromal cells (BMSCs)/beta-tricalcium phosphate (beta-TCP) composites have attracted a great deal of attention in bone tissue engineering. If more effective bone regeneration is to be achieved, efficient cell-seeding systems need to be clarified. In this study, we investigated the number of cells contained in composites, and the in vitro/vivo osteogenic differentiation capacity of composites using 4 conventional systems of seeding rat BMSCs into beta-TCP: soak, low-pressure, pipette, and syringe systems. The highest number of cells was contained in the composites from the syringe group. Moreover, after two-week osteogenic induction in vitro, the composites in the syringe group exhibited the highest osteogenic potential, which continued at 8 weeks after subcutaneous implantation in vivo. Our results indicated that efficient and appropriate cell-seeding could improve in vitro/vivo bone formation in composites and thus make a potential clinical contribution to successful bone tissue engineering.

Structure analysis of short peptides by analytical ultracentrifugation: Review.

Short peptides are potential drug candidates for pharmaceutical and biotech industries. Short peptides are natural ligands for numerous G-protein coupled receptors (GPCR) and hence constitute a large number of drug candidates. Synthetic short peptides are also extensively developed as agonistic or antagonistic ligands that function in a similar manner to antibodies, soluble receptors and protein ligands. Characterization of the peptides in solution is often performed in the presence of organic solvents, which can presumably generate the structure bound to the target surface and also enhance the solubility of the peptides. Analytical ultracentrifugation (AUC) technique should provide information on the state of self-association of the peptide in solution. Its application for short peptides has been far less than the applications for proteins. We believe that AUC should be used to show the associated state of the peptides, as reviewed in this paper.

Osteogenic activity of human fracture haematoma-derived progenitor cells is stimulated by low-intensity pulsed ultrasound in vitro.

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures.

Multifunction image intensifier for image processing.

A specially designed image intensifier tube for optical image processing has been developed. This tube can achieve the many types of processing important for pattern recognition such as deflection, zooming, gating, rotation, intensification, and logic operation in a single device. Experimental demonstrations are presented. Magnetic rotation of a pattern is proposed. The tube can perform the operations even in low level or incoherent light. Since processing is performed electrically or magnetically with no scanning, the processing speed is extremely fast.

Multiple mechanisms underlie neurotoxicity by different types of Alzheimer's disease mutations of amyloid precursor protein.

We examined a neuronal cell system in which single-cell expression of either familial Alzheimer's disease (FAD) gene V642I-APP or K595N/M596L-APP (NL-APP) in an inducible plasmid was controlled without affecting transfection efficiency. This system revealed that (i) low expression of both mutants exerted toxicity sensitive to both Ac-DEVD-CHO (DEVD) and glutathione ethyl ester (GEE), whereas wild-type APP (wtAPP) only at higher expression levels caused GEE/DEVD-resistant death to lesser degrees; (ii) toxicity by the V642I mutation was entirely GEE/DEVD sensitive; and (iii) toxicity by higher expression of NL-APP was GEE/DEVD resistant. The GEE/DEVD-sensitive death was sensitive to pertussis toxin and was due to G(o)-interacting His(657)-Lys(676) domain. The GEE/DEVD-resistant death was due to C-terminal Met(677)-Asn(695). APP mutants lacking either domain unraveled elaborate intracellular cross-talk between these domains. E618Q-APP, responsible for non-AD type of a human disease, only exerted GEE/DEVD-resistant death at higher expression. Therefore, (i) different FAD mutations in APP cause neuronal cell death through different cytoplasmic domains via different sets of mechanisms; (ii) expression levels of FAD genes are critical in activating specific death mechanisms; and (iii) toxicity by low expression of both mutants most likely reflects the pathogenetic mechanism of FAD.

A novel interferon-inducible gene expressed during myeloid differentiation.

The acute promyelocytic leukemia cell line, NB4, can be induced to differentiate to mature granulocytes by retinoic acid treatment. A novel retinoic acid-inducible cDNA clone, designated RI58, was isolated from a cDNA library constructed from retinoic acid-treated NB4 cells by differential hybridization. RI58 cDNA encodes a protein of 58kDa which has a similarity in its amino acids sequence to interferon (IFN)-inducible proteins. In addition, RI58 was induced by recombinant human IFN-alpha (rhIFN-alpha) in NB4 cells. RI58 was detectable within 4 hours post-stimulation with rhIFN-alpha, while it took as long as 1day after retinoic acid stimulation. Culture supernatant from retinoic acid-treated NB4 cells also induced RI58 expression similarly as rhIFN-alpha. This activity in culture supernatant was inhibited by anti-leukocyte IFN antiserum which showed specific reactivity to rhIFN-alpha. These results indicate that RI58 is induced by retinoic acid stimulation through autocrinally secreted IFN-alpha from NB4 cells. In the retinoic acid-treated NB4 cells, the expression of RI58 was increased along the process of differentiation. On the other hand, it was expressed constitutively in untreated non-hematopoietic cell lines and mature hematopoietic cell lines.

Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein.

We report a novel gene, designated Humanin (HN) cDNA, that suppresses neuronal cell death by K595N/M596L-APP (NL-APP), a mutant causing familial Alzheimer's disease (FAD), termed Swedish mutant. Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP. HN suppressed neurotoxicity by Abeta1-43 in the absence of N2 supplement, but could not inhibit Abeta secretion from NL-APP. HN could also protect neuronal cells from death by NL-APP lacking the 41st and 42nd residues of the Abeta region. Therefore, HN suppressed neuronal cell death by NL-APP not through inhibition of Abeta42 secretion, but with two targets for its inhibitory action: (i) the intracellular toxic mechanism directly triggered by NL-APP and (ii) neurotoxicity by Abeta. HN will contribute to the development of curative therapy of AD, especially as a novel reagent that could mechanistically supplement Abeta-production inhibitors.

Neuronal cell apoptosis by a receptor-binding domain peptide of ApoE4, not through low-density lipoprotein receptor-related protein.

Since an apolipoprotein E4 (ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [ApoE4(141-149)(2) or ApoE(141-155)(2)] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether ApoE4(141-149)(2)-induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), ApoE4(141-149)(2) also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. ApoE4(141-149)(2)-induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that ApoE4(141-149)(2) is able to exert neurotoxicity without involving LRP.