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Background: The tau protein phosphorylated at Thr181 (p-tau181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer’s disease (AD). Increased p-tau181 levels correlate well with amyloid-β (Aβ) pathology and precede neurofibrillary tangle formation in the early stage of AD; however, the relationship between p-tau181 and Aβ-mediated pathology is less well understood. We recently reported that p-tau181 represents axonal abnormalities in mice with Aβ pathology (AppNLGF). However, from which neuronal subtype(s) these p-tau181-positive axons originate remains elusive. Objective: The main purpose of this study is to differentiate neuronal subtype(s) and elucidate damage associated with p-tau181-positive axons by immunohistochemical analysis of AppNLGF mice brains. Methods: Colocalization between p-tau181 and (1) unmyelinated axons positive for vesicular acetylcholine transporter or norepinephrine transporter and (2) myelinated axons positive for vesicular glutamate transporter, vesicular GABA transporter, or parvalbumin in the brains of 24-month-old AppNLGF and control mice without Aβ pathology were analyzed. The density of these axons was also compared. Results: Unmyelinated axons of cholinergic or noradrenergic neurons did not overlap with p-tau181. By contrast, p-tau181 signals colocalized with myelinated axons of parvalbumin-positive GABAergic interneurons but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, whereas that of glutamatergic, GABAergic, or p-tau181-positive axons was less affected. Instead, myelin sheaths surrounding p-tau181-positive axons were significantly reduced in AppNLGF mice. Conclusion: This study demonstrates that p-tau181 signals colocalize with axons of parvalbumin-positive GABAergic interneurons with disrupted myelin sheaths in the brains of a mouse model of Aβ pathology.

The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1+ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1+ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1+ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.

Declines in mitochondrial functions are associated with aging. The combination of 5‐aminolevulinic acid (5‐ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5‐ALA and SFC (5‐ALA/SFC) on the healthspan and life span of Drosophila melanogaster. Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5‐ALA/SFC. Locomotor functions, life span, muscle architecture, and age‐associated changes in mitochondrial function were analyzed. We found that feeding 5‐ALA/SFC mitigated age‐associated declines in locomotor functions and extended organismal life span. Moreover, 5‐ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5‐ALA phosphate/SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age‐related declines in muscle functions, prevent age‐associated clinical conditions such as frailty, and extend healthspan and life span., Age‐related declines in physiological systems cause problematic features such as frailty. In this study, we found that 5‐aminolevulinic acid hydrochloride and sodium ferrous citrate (5‐ALA/SFC) mitigated age‐associated declines in locomotor functions and the muscle architecture, preserved the mitochondrial membrane potential, and extended life span in Drosophila melanogaster. 5‐ALA/SFC might be used to prevent age‐associated clinical conditions.

Phospho-tau 217, phospho-tau 231 and phospho-tau 181 in cerebrospinal fluid and plasma are promising biomarkers for the diagnosis of Alzheimer’s disease. All these p-tau proteins are detected in neurofibrillary tangles in brains obtained post-mortem from Alzheimer’s disease patients. However, increases in p-tau levels in cerebrospinal fluid and plasma during the preclinical stage of Alzheimer’s disease correlate with amyloid-β burden and precede neurofibrillary tangles in brains, suggesting that these p-tau proteins are indicative of amyloid-β-mediated brain pathology. In addition, phospho-tau 217 has greater sensitivity than phospho-tau 181, though it is unclear whether each of these p-tau variants contributes to the same or a different type of neuropathology prior to neurofibrillary tangle formation. In this study, we evaluated the intracerebral localization of p-tau in App knock-in mice with amyloid-β plaques without neurofibrillary tangle pathology (AppNLGF), in App knock-in mice with increased amyloid-β levels without amyloid-β plaques (AppNL) and in wild-type mice. Immunohistochemical analysis showed that phospho-tau 217 and phospho-tau 231 were detected only in AppNLGF mice as punctate structures around amyloid-β plaques, overlapping with the tau pathology marker, AT8 epitope phospho-tau 202/205/208. Moreover, phospho-tau 217 and phospho-tau 202/205/208 colocalized with the postsynaptic marker PSD95 and with a major tau kinase active, GSK3β. In contrast and similar to total tau, phospho-tau 181 signals were readily detectable as fibre structures in wild-type and AppNL mice and colocalized with an axonal marker neurofilament light chain. In AppNLGF mice, these phospho-tau 181-positive structures were disrupted around amyloid-β plaques and only partially overlapped with phospho-tau 217. These results indicate that phospho-tau 217, phospho-tau 231 and a part of phospho-tau 181 signals are markers of postsynaptic pathology around amyloid-β plaques, with phospho-tau 181 also being a marker of axonal abnormality caused by amyloid-β burden in brains.

Background: The locus coeruleus (LC), a brainstem nucleus comprising noradrenergic neurons, is one of the earliest regions affected by Alzheimer’s disease (AD). Amyloid-β (Aβ) pathology in the cortex in AD is thought to exacerbate the age-related loss of LC neurons, which may lead to cortical tau pathology. However, mechanisms underlying LC neurodegeneration remain elusive. Objective: Here, we aimed to examine how noradrenergic neurons are affected by cortical Aβ pathology in AppNL-G-F/NL-G-F knock-in mice. Methods: The density of noradrenergic axons in LC-innervated regions and the LC neuron number were analyzed by an immunohistochemical method. To explore the potential mechanisms for LC degeneration, we also examined the occurrence of tau pathology in LC neurons, the association of reactive gliosis with LC neurons, and impaired trophic support in the brains of AppNL-G-F/NL-G-F mice. Results: We observed a significant reduction in the density of noradrenergic axons from the LC in aged AppNL-G-F/NL-G-F mice without neuron loss or tau pathology, which was not limited to areas near Aβ plaques. However, none of the factors known to be related to the maintenance of LC neurons (i.e., somatostatin/somatostatin receptor 2, brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) were significantly reduced in AppNL-G-F/NL-G-F mice. Conclusion: This study demonstrates that cortical Aβ pathology induces noradrenergic neurodegeneration, and further elucidation of the underlying mechanisms will reveal effective therapeutics to halt AD progression.

The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1

Study question In the management of male infertility, we investigated whether urological consultation could improve the live birth rate, and who should visit urologists in the setting of IVF clinic. Summary answer Urologic consultation resulted in improvement of semen quality and live birth rate with more IVF use in those with adverse semen parameters. What is known already Male factor infertility exists in about a half of infertility couples. This accounts for about 8% in male reproductive age. Therefore, ideally every male partner of infertility couples attempting conception should have a urological evaluation. However, it is not very easy to access urologists who specialized in reproductive medicine in Japan because we have very few of such urologists. One the other hand, a certain number of couples are wasting their time during IVF failure without urological evaluation. Study design, size, duration This is a single-institution retrospective study. We enrolled male partners of infertility couples who visited Kameda IVF clinic Makuhari, Chiba, Japan, between May 2016 and December 2020 and followed at least one year. Live birth rate and the frequency of IVF use were investigated according to semen quality and urological consultation status. Chi-square tests and T tests were used to compare the results between groups. Participants/materials, setting, methods Among 2225 couples who visited Kameda IVF clinic Makuhari, 803 male partners (Group A, 36.0%) were evaluated by urologists who were specialized in male reproductive medicine. Remaining 1422 patients did not (Group B, 64.0%). Lifestyle evaluation, physical examination, semen analyses, scrotal ultrasonography, blood test including sexual hormones and zinc concentration were performed in Group A. Semen analyses and lifestyle evaluation were performed in Group B. Urological treatments were done according to factors of male infertility. Main results and the role of chance Semen quality was worse in Group A as compared to Group B (sperm motility, 28.5±16.9% vs. 46.0±17.0%; total sperm count, 105±108 million/mL vs. 176±155; total motile sperm count, 34±49 vs.87±98; mean±S.D.; p = 0.0001, 0.0001, 0.0001, A vs. B, respectively). After urologic consultation and managements, sperm motility was improved to 34±18% (p = 0.001). Live birth rate in groups A and B were similar (56.0% vs. 57.2%), however couples who obtained a child in Group A used IVF more often than those in Group B (70% vs. 49.9%, p 50 million, n = 900), 119 visited urologist (31.1%, Group 3) and 781 did not (Group 4). Live birth rate and the use of IVF were not different between Groups 3 and 4 (51.1% vs.60.9%; 50.4% vs. 62.9%). Limitations, reasons for caution This study is a single-institution, retrospective study in the setting of IVF clinic. There may be a selection bias since men first visit gynecologists. These could affect the study results. Wider implications of the findings In the setting of IVF clinic, urologic consultation resulted in improved semen quality and better live birth rate with the use of IVF, especially in those who have adverse semen parameters. The results of this study encourage patients to see urologists and physicians to introduce urologist to patients. Trial registration number not applicable

The molecular biological mechanisms of Alzheimer’s disease (AD) involve disease-associated crosstalk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, including Rac1, as key players in PDN collapse. Using publicly available as well as our own gene expression data, we confirmed that the mRNA expression level of the RAC1 gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilized Drosophila as a genetic model and found that modest knockdown of Rac1 in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator of RAC1 in AD brains. As the Braak neurofibrillary tangle (NFT) stage progressed, the expression levels of hsa-miR-101-3p were increased specifically in the EC. Furthermore, overexpression of hsa-miR-101-3p in the human neuronal cell line SH-SY5Y caused RAC1 downregulation. These results highlight the utility of our integrated network approach for identifying causal changes leading to neurodegeneration in AD.

Hyperphosphorylation of the microtubule-associated protein tau is associated with many neurodegenerative diseases, including Alzheimer’s disease. Microtubule affinity-regulating kinases (MARK) 1–4 and cyclin-dependent kinase 5 (Cdk5) are tau kinases under physiological and pathological conditions. However, their functional relationship remains elusive. Here, we report a novel mechanism by which Cdk5 activates MARK4 and augments tau phosphorylation, accumulation and toxicity. MARK4 is highly phosphorylated at multiple sites in the brain and in cultured neurons, and inhibition of Cdk5 activity reduces phosphorylation levels of MARK4. MARK4 is known to be activated by phosphorylation at its activation loop by liver kinase B1 (LKB1). In contrast, Cdk5 increased phosphorylation of MARK4 in the spacer domain, but not in the activation loop, and enhanced its kinase activity, suggesting a novel mechanism by which Cdk5 regulates MARK4 activity. We also demonstrated that co-expression of Cdk5 and MARK4 in mammalian cultured cells significantly increased the levels of tau phosphorylation at both Cdk5 target sites (SP/TP sites) and MARK target sites (Ser262), as well as the levels of total tau. Furthermore, using a Drosophila model of tau toxicity, we demonstrated that Cdk5 promoted tau accumulation and tau-induced neurodegeneration via increasing tau phosphorylation levels at Ser262 by a fly ortholog of MARK, Par-1. This study suggests a novel mechanism by which Cdk5 and MARK4 synergistically increase tau phosphorylation and accumulation, consequently promoting neurodegeneration in disease pathogenesis.

The locus coeruleus (LC) is the noradrenaline (norepinephrine, NE)-containing nucleus in the brainstem and innervates into widespread brain regions. This LC-NE system plays a critical role in a variety of brain functions, including attention, arousal, emotion, cognition, and the sleep-wake cycle. The LC is one of the brain regions vulnerable to the occurrence of neurofibrillary tangles (NFTs), which is associated with "primary age-related tauopathy (PART)" that describes the pathology commonly observed in the brains of aged individuals. In Alzheimer's disease (AD), the LC is one of the first places to develop NFTs, which may act as a seed for subsequent spreading of the pathology throughout the brain upon amyloid-β (Aβ) accumulation. As AD progresses, significant neuron loss occurs in the LC. Moreover, LC neurodegeneration is not only a consequence of AD, but also drives clinical and pathological manifestations of AD, such as microglial dysregulation, sleep disturbance, cognitive decline, and neurovascular dysfunction. Therefore, prevention of NFT pathology and neuron loss in the LC-NE system is critical for suppressing the progression of AD. We propose that targeting aging itself may be a proactive intervention against age-associated changes in the LC. Such an approach could open the way for novel interventions against age-associated neurodegenerative disorders, in particular, AD.

For decades, the fruit fly

Introduction Late-onset hypogonadism is associated with higher mortality largely due to cardiovascular disease (CVD). Objective We investigated whether a low plasma free testosterone (FT) level could predict cardiovascular events in middle-aged Japanese men with coronary risk factors. Methods We reviewed retrospectively our institutional database of patients with any coronary risk factor (hypertension, diabetes, dyslipidemia, smoking, and obesity) without a previous history of CVD. Serum early morning free testosterone (FT) levels, collected at baseline, were analyzed. Patient age, baseline FT level, and the above coronary risk factor were selected as potential predictors of cardiovascular events. Multivariable time-to-event analysis (Cox proportional hazards), and Kaplan-Meier curve were performed to define impact of the FT level on time of cardiovascular (CV) events. Results Four hundred and sixty-six male outpatients (37–89 years old, mean ± SD = 65.1 ± 10.5 years) who had any coronary risk without a previous history of CVD were accumulated. During the mean follow-up period of 84.8 months, a total of 126 CV events occurred. Kaplan–Meier survival analysis by tertile of plasma FT levels revealed that the subjects with the lowest FT tertiles (9.3 pg/ml) (P9.3 pg/ml) and those with the second tertiles of plasma FT (6.6–9.2 pg/ml) had significantly lower CV event risk compared to those with the lowest FT tertiles ( Conclusion In middle-aged Japanese men with some coronary risk factors, lower FT is a predictor for the development of CVDs, independently on any coronary risk factors and age. Disclosure Work supported by industry: no.

SummaryBrain neurons play a central role in organismal aging, but there is conflicting evidence about the roles of neuronal glucose availability, glucose uptake and metabolism in aging and extended lifespan. Here, we analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically-encoded fluorescent ATP biosensor, we found decreased ATP concentration in the neuronal somata of aged flies, which was correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to reductions in ATP. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed age-dependent locomotor deficits and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest lifespans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging.

Accumulation of the microtubule-associated protein tau is associated with Alzheimer’s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser262 and Ser356 play critical roles in tau accumulation and toxicity. Microtubule-affinity regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a doublede novomutation in the linker region of MARK4, ΔG316E317InsD, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4ΔG316E317Dincreases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser262/356-dependent and -independent mechanisms. Using transgenicDrosophilaexpressing human MARK4 (MARK4wt) or a mutant version of MARK4 (MARK4ΔG316E317D), we found that co-expression of MARK4wtand MARK4ΔG316E317Dincreased total tau levels and enhanced tau-induced neurodegeneration, and that MARK4ΔG316E317Dhad more potent effects than MARK4wt. Interestingly, thein vitrokinase activities of MARK4wtand MARK4ΔG316E317Dwere similar. Blocking tau phosphorylation at Ser262 and Ser356 by alanine substitutions protected tau from the effects of MARK4wt, but not from MARK4ΔG316E317D. While both MARK4wtand MARK4ΔG316E317Dincreased the levels of oligomeric forms of tau, MARK4ΔG316E317Dfurther boosted the levels of tau phosphorylated at several sites other than Ser262/356 and increased the detergent insolubility of tauin vivo. Together, these findings suggest that MARK4ΔG316E317Dincreases tau levels and exacerbates tau toxicity via an additional gain-of-function mechanism, and that modification in this region of MARK4 may impact disease pathogenesis.

Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4(ΔG316E317D) increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4(wt)) or a mutant version of MARK4 (MARK4(ΔG316E317D)), we found that coexpression of MARK4(wt) and MARK4(ΔG316E317D) increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4(ΔG316E317D) had more potent effects than MARK4(wt). Interestingly, the in vitro kinase activities of MARK4(wt) and MARK4(ΔG316E317D) were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4(wt) did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4(ΔG316E317D) did. Both MARK4(wt) and MARK4(ΔG316E317D) increased the levels of oligomeric forms of tau; however, only MARK4(ΔG316E317D) further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4(ΔG316E317D) increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Background Cerebral amyloidosis, neuroinflammation, and tauopathy are key features of Alzheimer’s disease (AD), but interactions among these features remain poorly understood. Our previous multiscale molecular network models of AD revealed TYROBP as a key driver of an immune- and microglia-specific network that was robustly associated with AD pathophysiology. Recent genetic studies of AD further identified pathogenic mutations in both TREM2 and TYROBP. Methods In this study, we systematically examined molecular and pathological interactions among Aβ, tau, TREM2, and TYROBP by integrating signatures from transgenic Drosophila models of AD and transcriptome-wide gene co-expression networks from two human AD cohorts. Results Glial expression of TREM2/TYROBP exacerbated tau-mediated neurodegeneration and synergistically affected pathways underlying late-onset AD pathology, while neuronal Aβ42 and glial TREM2/TYROBP synergistically altered expression of the genes in synaptic function and immune modules in AD. Conclusions The comprehensive pathological and molecular data generated through this study strongly validate the causal role of TREM2/TYROBP in driving molecular networks in AD and AD-related phenotypes in flies. Electronic supplementary material The online version of this article (10.1186/s13073-018-0530-9) contains supplementary material, which is available to authorized users.

Summary For decades, the fruit fly Drosophila melanogaster has been an efficient genetic model to investigate many aspects of human neurodegenerative diseases. Through genetic and pharmacologic approaches, these studies have revealed the molecular mechanisms underlying disease pathogenesis and provided therapeutic implications. Here, we describe a protocol for assessing Alzheimer's disease-related amyloid-β toxicity in a transgenic fly model through biochemical, histological, and behavioral analyses. We also discuss the advantages and limitations of our protocols. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) .

Abnormal deposition of the microtubule-associated protein tau is a common pathological feature of multiple neurodegenerative diseases, including Alzheimer's disease (AD), and plays critical roles in their pathogenesis. Disruption of calcium homeostasis and the downstream kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) coincides with pathological phosphorylation of tau in AD brains. However, it remains unclear whether and how dysregulation of CaMKII affects tau toxicity. Using a Drosophila model, we found that CaMKII promotes neurodegeneration caused by tau phosphorylated at the AD-associated sites Ser262/356. Overexpression of CaMKII promoted, while RNA-mediated knockdown of CaMKII and inhibition of CaMKII activity by expression of an inhibitory peptide suppressed, tau-mediated neurodegeneration. Blocking tau phosphorylation at Ser262/356 by alanine substitutions suppressed promotion of tau toxicity by CaMKII, suggesting that tau phosphorylation at these sites is required for this phenomenon. However, neither knockdown nor overexpression of CaMKII affected tau phosphorylation levels at Ser262/356, suggesting that CaMKII is not directly involved in tau phosphorylation at Ser262/356 in this model. These results suggest that a pathological cascade of events, including elevated levels of tau phosphorylated at Ser262/356 and aberrant activation of CaMKII, work in concert to promote tau-mediated neurodegeneration.

Three different partners, ENEA, JAEA ed University of Tokyo, have been involved during 2014-2015 in the Round Robin experimentation for the assessment of the soundness of the analitycal procedure for the determination of the Nitrogen impurities contained inside a solid Lithium sample. Two different kinds of Lithium samples, differing by about an order of magnitude in Nitrogen concentration (∼230 wppm; ∼20-30 wppm), have been selected for this cross analysis. The agreement of the achieved results appears very good for what concerns the most concentrated Lithium and indicates each partner's procedure is appropriate and intrinsecally able to lead to meaningful values, characterized by a relative uncertainty of just few %. The smaller agreement in the case of the less concentrated Lithium anyway points out that particular attention must be paid to reduce as much as possible any source of external contamination and highlights the importance of the proper blank subtraction. © 2016 EURATOM-ENEA.

SUMMARYTo study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimer’s Disease (LOAD), we performed an integrative network analysis of whole-genome DNA and RNA sequencing profiling of four cortical areas, including the parahippocampal gyrus, across 364 donors spanning the full spectrum of LOAD-related cognitive and neuropathological disease severities. Our analyses revealed thousands of molecular changes and uncovered for the first-time multiple neuron specific gene subnetworks most dysregulated in LOAD. ATP6V1A, a critical subunit of vacuolar-type H+-ATPase (v-ATPase), was predicted to be a key regulator of one neuronal subnetwork and its role in disease-related processes was evaluated through CRISPR-based manipulation of human induced pluripotent stem cell derived neurons and RNAi-based knockdown in transgenic Drosophila models. This study advances our understanding of LOAD pathogenesis by providing the global landscape and detailed circuits of complex molecular interactions and regulations in several key brain regions affected by LOAD and the resulting network models provide a blueprint for developing next generation therapeutics against LOAD.

The molecular biological mechanisms of Alzheimer’s disease (AD) involve disease-associated cross-talk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, includingRac1, as key players in PDN collapse. Using publicly available gene expression data, we confirmed that the mRNA expression level of theRAC1gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilizedDrosophilaas a genetic model and found that modest knockdown ofRac1in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator ofRAC1in AD brains. As the Braak neurofibrillary tangle (NFT) stage progressed, the expression levels of hsa-miR-101-3p were upregulated specifically in the EC. Furthermore, overexpression of hsa-miR-101-3p in the human neuronal cell line SH-SY5Y causedRAC1downregulation. These results highlight the utility of our integrated network approach for identifying causal changes leading to neurodegeneration in AD.

We report on a new in situ laser spectroscopy technique using superfluid helium and a highly energetic ion beam. In taking advantage of superfluid helium as a host matrix for laser spectroscopy, we were able to develop an extremely low background detection system. The first demonstration using Rb-85(31+) ion beam (66 MeV/nucleon) accelerated at the RIKEN accelerator facility shows improvement of detection limit by two orders of magnitude. A quantitative evaluation of Rb atoms in the observation region enables an analysis of their optical absorption cross-section in superfluid helium from the fluorescence intensity dependence on ion-beam intensity and applied laser power. (C) 2018 The Japan Society of Applied Physics

Summary Brain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. Here, we analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, we found decreased ATP concentration in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging., Graphical Abstract For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.isci.2020.101979, Highlights • Imaging of Drosophila brain reveals aged neurons suffer from energy deficits • Increased neuronal glucose uptake attenuates age-dependent declines in ATP • Increased glucose uptake is beneficial despite age-dependent mitochondrial damage • Increased neuronal glucose uptake and dietary restriction further extend life span, Biological Sciences; Cellular Neuroscience; Endocrinology; Neuroscience; Physiology

Background Alzheimer’s disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Mouse models of Aβ amyloidosis generated by knock-in (KI) of a humanized Aβ sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aβ pathology observed in AD: the Swedish (NL) mutation, which elevates total Aβ production; the Beyreuther/Iberian (F) mutation, which increases the Aβ42/Aβ40 ratio; and the Arctic (G) mutation, which promotes Aβ aggregation. AppNL-G-F mice harbor all three mutations and develop progressive Aβ amyloidosis and neuroinflammatory response in broader brain areas, whereas AppNL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aβ pathology, we assessed emotional and cognitive domains of AppNL-G-F and AppNL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks. Results Assessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, AppNL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, AppNL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both AppNL/NL and AppNL-G-F/NL-G-F mice exhibited intact learning and memory up to 15–18 months of age, whereas AppNL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, AppNL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions. Conclusion AppNL/NL and AppNL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that AppNL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration. Electronic supplementary material The online version of this article (10.1186/s12868-018-0446-8) contains supplementary material, which is available to authorized users.

Objective Involvement of the hypothalamus is rare in patients with systemic lupus erythematosus (SLE). In this study, we measured cerebrospinal fluid (CSF) orexin-A levels in SLE patients with hypothalamic lesions to investigate whether the orexin system plays a role in SLE patients with hypothalamic lesions who present with excessive daytime sleepiness (EDS). Methods Orexin-A levels were measured in CSF from four patients with SLE who presented with hypothalamic lesions detected by MRI. Three patients underwent repeated CSF testing. All patients met the updated American College of Rheumatology revised criteria for SLE. Results Tests for serum anti-aquaporin-4 antibodies, CSF myelin basic protein and CSF oligoclonal bands were negative in all patients. All patients presented with EDS. Low to intermediate CSF orexin-A levels (92–180 pg/ml) were observed in three patients in the acute stage, two of whom (patients 1 and 2) underwent repeated testing and showed increased CSF orexin-A levels, reduced abnormal hypothalamic lesion intensities detected by MRI and EDS dissipation at follow-up. In contrast, CSF orexin-A levels were normal in one patient (patient 4) while in the acute stage and at follow-up, despite improvements in EDS and MRI findings. Patient 4 showed markedly increased CSF interleukin-6 levels (1130 pg/ml) and a slightly involved hypothalamus than the other patients. Conclusions Our findings suggest that the orexinergic system has a role in EDS in SLE patients with hypothalamic lesions. Furthermore, cytokine-mediated tissue damage might cause EDS without orexinergic involvement.

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. In this study, we sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. Our results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

Background: Fingolimod is approved for the treatment of relapsing-remitting multiple sclerosis and was effective in experimental autoimmune neuritis in rats, a possible model for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We aimed to evaluate the efficacy of fingolimod in delaying disability progression in patients with CIDP who withdrew from currently effective treatments (intravenous immunoglobulin [IVIg] or corticosteroids). Methods: This double-blind, multicentre, randomised, placebo-controlled, parallel-group, event-driven study was done at 48 neurology centres in Australia, Canada, Israel, Japan, the USA, and nine countries in Europe. Participants with CIDP who were receiving IVIg or corticosteroids were randomly assigned (1:1) to once-daily oral fingolimod 0·5 mg or placebo. Owing to the event-driven design, treatment duration was flexible and could be up to 4·5 years. Randomisation was done with an automated interactive voice response-web response system and was stratified by Inflammatory Neuropathy Cause and Treatment (INCAT) disability scale scores. Previous IVIg treatment was discontinued after one final course ending the day before the first dose of fingolimod or placebo was given, whereas corticosteroids were tapered off over 8 weeks after randomisation. The primary endpoint was time to first confirmed worsening (≥1 point increase on the adjusted INCAT disability scale score versus baseline) and was assessed in the full analysis set, which consisted of all patients who underwent randomisation and had at least one efficacy assessment for the primary analysis. The survival distribution functions of time to first worsening were estimated within each treatment group according to the Kaplan-Meier survival distribution function and compared with a stratified log-rank test. The trial is registered with ClinicalTrials.gov, number NCT01625182. Findings: Of 106 participants randomly assigned between Jan 24, 2013, and March 10, 2016, 54 received fingolimod (41 who had been receiving IVIg and 13 who had been receiving corticosteroids) and 52 received placebo (41 who had been receiving IVIg and 11 who had been receiving corticosteroids). The trial ended for futility as recommended by an independent data monitoring committee after an interim analysis when 44 confirmed worsening events had occurred. At the end of the study, the survival estimate of the proportion of participants free from confirmed worsening was not significantly different between the fingolimod group (42%, 95% CI 23–60) and the placebo group (43%, 28–59; p=0·91). Adverse events occurred in 41 (76%) participants receiving fingolimod and 44 (85%) on placebo, and serious adverse events occurred in nine (17%) and four (8%) patients, respectively. The most common adverse events with fingolimod were headache (12 [22%] patients), hypertension (ten [19%]), and extremity pain (seven [13%]). Adverse events leading to study discontinuation occurred in seven (13%) participants on fingolimod and none on placebo. Interpretation: Fingolimod 0·5 mg once-daily was not better than placebo for the treatment of CIDP. Future trial designs should take account of the possibility that if IVIg is stopped abruptly, some patients might relapse soon afterwards whereas others might remain in remission. Funding: Novartis Pharma. © 2018 Elsevier Ltd

As the vehicle for communicating the intended biomechanical regimen to the tooth undergoing a treatment, brackets occupy a central place in the orthodontic armamentarium. The evolution and types of brackets will be presented in this chapter, along with the different types of materials and fabrication procedures employed for the brackets. An important feature will be an extensive discussion of reports on clinical performance. General structure-property relationships for the bracket materials have been previously presented in Chapters 1–3 Chapter 1 Chapter 2 Chapter 3.

Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer's disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains.

Additional file 1: Figure S1. Preparation of donut-shaped ice-block of culture medium. Figure S2. Validation of vimentin structures after low temperature treatment. Figure S3. Validation of vimentin amount after low temperature treatment. Figure S4. Stiffness of cells measured during nanoneedle insertion at different temperatures.

We performed dielectric measurements on thermally aged carbon-black-filled chloroprene rubber (CR). Primary and secondary processes were observed in unaged and thermally aged CR. The primary process is due to the cooperative motion of chloroprene chains in CR, and the secondary process is due to the local motion of chloroprene chains. For the primary process, the relaxation time slightly increases and symmetric shape parameter of the loss spectrum decreases with increasing aging time. For the secondary process, the relaxation strength decreases with increasing aging time, and reaches zero after 50 h. These changes in the dielectric relaxation parameters are due to the progress of the dehydrochlorination reaction at the chloroprene chains.

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimer's disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic β-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. Here, we investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases., Author summary Wolfram syndrome (WS), a neurodegenerative disorder with an autosomal recessive inheritance pattern, has a variable clinical presentation that includes diabetes mellitus, optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. Homozygous mutations in WFS1 are causative for WS. The prognosis of WS is poor, and most patients die prematurely with respiratory failure due to brain stem atrophy. However, the mechanisms underlying the neurological manifestations of WS remain elusive. In this study, we used the fruit fly Drosophila to examine the neurological features of WS by generating genetically modified flies harboring knockdown of wfs1, the fly homolog of WFS1, in the central nervous system. These flies developed age-dependent behavioral deficits, neurodegeneration and premature death. wfs1-deficient flies were vulnerable to various age-related stressors such as oxidative stress and excitotoxicity, and to neurodegeneration caused by Alzheimer’s disease-related toxic proteins. The premature death phenotype in wfs1-deficient flies was ameliorated by administration of riluzole, which inhibits glutamate-induced excitotoxicity. This study provides insight into the mechanisms underlying neurodegeneration not only in WS, but also in age-associated neurodegenerative diseases such as Alzheimer’s disease.