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State-of-the-art Earth system models typically employ grid spacings of O(100 km), which is too coarse to explicitly resolve main drivers of the flow of energy and matter across the Earth system. In this paper, we present the new ICON-Sapphire model configuration, which targets a representation of the components of the Earth system and their interactions with a grid spacing of 10 km and finer. Through the use of selected simulation examples, we demonstrate that ICON-Sapphire can (i) be run coupled globally on seasonal timescales with a grid spacing of 5 km, on monthly timescales with a grid spacing of 2.5 km, and on daily timescales with a grid spacing of 1.25 km; (ii) resolve large eddies in the atmosphere using hectometer grid spacings on limited-area domains in atmosphere-only simulations; (iii) resolve submesoscale ocean eddies by using a global uniform grid of 1.25 km or a telescoping grid with the finest grid spacing at 530 m, the latter coupled to a uniform atmosphere; and (iv) simulate biogeochemistry in an ocean-only simulation integrated for 4 years at 10 km. Comparison of basic features of the climate system to observations reveals no obvious pitfalls, even though some observed aspects remain difficult to capture. The throughput of the coupled 5 km global simulation is 126 simulated days per day employing 21 % of the latest machine of the German Climate Computing Center. Extrapolating from these results, multi-decadal global simulations including interactive carbon are now possible, and short global simulations resolving large eddies in the atmosphere and submesoscale eddies in the ocean are within reach.

In the era of exascale computing, machines with unprecedented computing power are available. Making efficient use of these massively parallel machines, with millions of cores, presents a new challenge. Multi-level and multi-dimensional parallelism will be needed to meet this challenge. Coarse-grained component concurrency provides an additional parallelism dimension that complements typically used parallelization methods such as domain decomposition and loop-level shared-memory approaches. While these parallelization methods are data-parallel techniques, and they decompose the data space, component concurrency is a function-parallel technique, and it decomposes the algorithmic space. This additional dimension of parallelism allows us to extend scalability beyond the limits set by established parallelization techniques. It also offers a way to maintain performance (by using more compute power) when the model complexity is increased by adding components, such as biogeochemistry or ice sheet models. Furthermore, concurrency allows each component to run on different hardware, thus leveraging the usage of heterogeneous hardware configurations. In this work we study the characteristics of component concurrency and analyse its behaviour in a general context. The analysis shows that component concurrency increases the “parallel workload”, improving the scalability under certain conditions. These generic considerations are complemented by an analysis of a specific case, namely the coarse-grained concurrency in the multi-level parallelism context of two components of the ICON modelling system: the ICON ocean model ICON-O and the marine biogeochemistry model HAMOCC. The additional computational cost incurred by the biogeochemistry module is about 3 times that of the ICON-O ocean stand alone model, and data parallelization techniques (domain decomposition and loop-level shared-memory parallelization) present a scaling limit that impedes the computational performance of the combined ICON-O–HAMOCC model. Scaling experiments, with and without concurrency, show that component concurrency extends the scaling, in cases doubling the parallel efficiency. The experiments' scaling results are in agreement with the theoretical analysis.

Earth system and climate modelling involves the simulation of processes on a wide range of scales and within and across various compartments of the Earth system. In practice, component models are often developed independently by different research groups, adapted by others to their special interests and then combined using a dedicated coupling software. This procedure not only leads to a strongly growing number of available versions of model components and coupled setups but also to model- and high-performance computing (HPC)-system-dependent ways of obtaining, configuring, building and operating them. Therefore, implementing these Earth system models (ESMs) can be challenging and extremely time consuming, especially for less experienced modellers or scientists aiming to use different ESMs as in the case of intercomparison projects. To assist researchers and modellers by reducing avoidable complexity, we developed the ESM-Tools software, which provides a standard way for downloading, configuring, compiling, running and monitoring different models on a variety of HPC systems. It should be noted that ESM-Tools is not a coupling software itself but a workflow and infrastructure management tool to provide access to increase usability of already existing components and coupled setups. As coupled ESMs are technically the more challenging tasks, we will focus on coupled setups, always implying that stand-alone models can benefit in the same way. With ESM-Tools, the user is only required to provide a short script consisting of only the experiment-specific definitions, while the software executes all the phases of a simulation in the correct order. The software, which is well documented and easy to install and use, currently supports four ocean models, three atmosphere models, two biogeochemistry models, an ice sheet model, an isostatic adjustment model, a hydrology model and a land-surface model. Compared to previous versions, ESM-Tools has lately been entirely recoded in a high-level programming language (Python) and provides researchers with an even more user-friendly interface for Earth system modelling. ESM-Tools was developed within the framework of the Advanced Earth System Model Capacity project, supported by the Helmholtz Association.

Abstract This work documents the ICON‐Earth System Model (ICON‐ESM V1.0), the first coupled model based on the ICON (ICOsahedral Non‐hydrostatic) framework with its unstructured, icosahedral grid concept. The ICON‐A atmosphere uses a nonhydrostatic dynamical core and the ocean model ICON‐O builds on the same ICON infrastructure, but applies the Boussinesq and hydrostatic approximation and includes a sea‐ice model. The ICON‐Land module provides a new framework for the modeling of land processes and the terrestrial carbon cycle. The oceanic carbon cycle and biogeochemistry are represented by the Hamburg Ocean Carbon Cycle module. We describe the tuning and spin‐up of a base‐line version at a resolution typical for models participating in the Coupled Model Intercomparison Project (CMIP). The performance of ICON‐ESM is assessed by means of a set of standard CMIP6 simulations. Achievements are well‐balanced top‐of‐atmosphere radiation, stable key climate quantities in the control simulation, and a good representation of the historical surface temperature evolution. The model has overall biases, which are comparable to those of other CMIP models, but ICON‐ESM performs less well than its predecessor, the Max Planck Institute Earth System Model. Problematic biases are diagnosed in ICON‐ESM in the vertical cloud distribution and the mean zonal wind field. In the ocean, sub‐surface temperature and salinity biases are of concern as is a too strong seasonal cycle of the sea‐ice cover in both hemispheres. ICON‐ESM V1.0 serves as a basis for further developments that will take advantage of ICON‐specific properties such as spatially varying resolution, and configurations at very high resolution.

The German Bight was exposed to record high riverine discharges in June 2013, as a result of flooding of the Elbe and Weser rivers. Several anomalous observations suggested that the hydrodynamical and biogeochemical states of the system were impacted by this event. In this study, we developed a biogeochemical model and coupled it with a previously introduced high-resolution hydrodynamical model of the southern North Sea in order to better characterize these impacts and gain insight into the underlying processes. Performance of the model was assessed using an extensive set of in situ measurements for the period 2011–2014. We first improved the realism of the hydrodynamic model with regard to the representation of cross-shore gradients, mainly through inclusion of flow-dependent horizontal mixing. Among other characteristic features of the system, the coupled model system can reproduce the low salinities, high nutrient concentrations and low oxygen concentrations in the bottom layers observed within the German Bight following the flood event. Through a scenario analysis, we examined the sensitivity of the patterns observed during July 2013 to the hydrological and meteorological forcing in isolation. Within the region of freshwater influence (ROFI) of the Elbe–Weser rivers, the flood event clearly dominated the changes in salinity and nutrient concentrations, as expected. However, our findings point to the relevance of the peculiarities in the meteorological conditions in 2013 as well: a combination of low wind speeds, warm air temperatures and cold bottom-water temperatures resulted in a strong thermal stratification in the outer regions and limited vertical nutrient transport to the surface layers. Within the central region, the thermal and haline dynamics interactively resulted in an intense density stratification. This intense stratification, in turn, led to enhanced primary production within the central region enriched by nutrients due to the flood but led to reduction within the nutrient-limited outer region, and it caused a widespread oxygen depletion in bottom waters. Our results further point to the enhancement of the current velocities at the surface as a result of haline stratification and to intensification of the thermohaline estuarine-like circulation in the Wadden Sea, both driven by the flood event.

Introduction: Improved understanding of vestibulodynia pathophysiology is required to develop appropriately targeted treatments. Established features include vulvovaginal hyperinnervation, increased nociceptive signalling and hypersensitivity. Emerging evidence indicates macrophage-neuron signalling contributes to chronic pain pathophysiology. Macrophages are broadly classified as M1 or M2, demonstrating pro-nociceptive or anti-nociceptive effects respectively. This study investigates the impact of clodronate liposomes, a macrophage depleting agent, on nociceptive signalling in a mouse model of vestibulodynia., Methods: Microinjection of complete Freund's adjuvant (CFA) at the vaginal introitus induced mild chronic inflammation in C57Bl/6J mice. A subgroup was treated with the macrophage depleting agent clodronate. Control mice received saline. After 7 days, immunolabelling for PGP9.5, F4/80+CD11c+ and F4/80+CD206+ was used to compare innervation density and presence of M1 and M2 macrophages respectively in experimental groups. Nociceptive signalling evoked by vaginal distension was assessed using immunolabelling for phosphorylated MAP extracellular signal-related kinase (pERK) in spinal cord sections. Hyperalgesia was assessed by visceromotor response to graded vaginal distension., Results: CFA led to increased vaginal innervation (p < 0.05), increased pERK-immunoreactive spinal cord dorsal horn neurons evoked by vaginal-distension (p < 0.01) and enhanced visceromotor responses compared control mice (p < 0.01). Clodronate did not reduce vaginal hyperinnervation but significantly reduced the abundance of M1 and M2 vaginal macrophages and restored vaginal nociceptive signalling and vaginal sensitivity to that of healthy control animals., Conclusions: We have developed a robust mouse model of vestibulodynia that demonstrates vaginal hyperinnervation, enhanced nociceptive signalling, hyperalgesia and allodynia. Macrophages contribute to hypersensitivity in this model. Macrophage-sensory neuron signalling pathways may present useful pathophysiological targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Castro, Harrington, Chegini, Matusica, Spencer, Brierley, Haberberger and Barry.)

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI 25k , and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI 25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI 25k and Lipo 2K, respectively. Unlike PEI 25k , N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Aims: Detection and quantification of myocardial scars are helpful for diagnosis of heart diseases and for personalized simulation models. Scar tissue is generally characterized by a different conduction of excitation. We aim at estimating conductivity-related parameters from endocardial mapping data. Solving this inverse problem requires computationally expensive monodomain simulations on fine discretizations. We aim at accelerating the estimation by combining electrophysiology models of different complexity., Methods and Results: Distributed parameter estimation is performed by minimizing the misfit between simulated and measured electrical activity on the endocardial surface, subject to the monodomain model and regularization. We formulate this optimization problem, including the modelling of scar tissue and different regularizations, and design an efficient solver. We consider grid hierarchies and monodomain-eikonal model hierarchies in a recursive multilevel trust-region method. With numerical examples, efficiency and estimation quality, depending on the data, are investigated. The multilevel solver is significantly faster than a comparable single level solver. Endocardial mapping data of realistic density appears to be sufficient to provide quantitatively reasonable estimates of location, size, and shape of scars close to the endocardial surface., Conclusion: In several situations, scar reconstruction based on eikonal and monodomain models differ significantly, suggesting the use of the more involved monodomain model for this purpose. Eikonal models can accelerate the computations considerably, enabling the use of complex electrophysiology models for estimating myocardial scars from endocardial mapping data., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Background: Alzheimer's disease (AD) pathology consists of intraneuronal neurofibrillary tangles, made of hyperphosphorylated tau and extracellular accumulation of beta amyloid (Aβ) in Aβ plaques. There is an extensive debate as to which pathology initiates and is responsible for cellular loss in AD., Methods: Using confocal and light microscopy, post mortem brains from control and AD cases, an antibody to SOD2 as a marker for mitochondria and an antibody to all forms of tau, we analyzed mitochondrial density in tau positive neurons along with nuclear degradation by calculating the raw integrative density., Results: Our findings showed an extensive staining of aggregated tau in cell bodies, dystrophic neurites and neurofilaments in AD with minimal staining in control tissue, along with a marked decrease in mitochondria in tau positive (tau+) neurons. The control or tau negative (tau-) neurons in AD contained an even distribution of mitochondria, which was greatly diminished in tau+ neurons by 40%. There were no significant differences between control and tau- neurons in AD. Tau+ neurons showed marked nuclear degradation which appeared to progress with the extent of tau aggregation. The aggregated tau infiltrated and appeared to break the nuclear envelope with progressively more DNA exiting the nucleus and associating with the aggregated intracellular tau., Conclusion: We report that the mitochondrial decrease is likely due to a decrease in the protein synthesis rather than a redistribution of mitochondria because of the decreased axonal transport. We suggest that the decrease in mitochondria and nuclear degradation are key mechanisms for the neuronal loss seen in AD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Neuronal loss in specific brain regions and neurons with intracellular inclusions termed Lewy bodies are the pathologic hallmark in both Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Lewy bodies comprise of aggregated intracellular vesicles and proteins and α-synuclein is reported to be a major protein component. Using human brain tissue from control, PD and DLB and light and confocal immunohistochemistry with antibodies to superoxide dismutase 2 as a marker for mitochondria, α-synuclein for Lewy bodies and βIII Tubulin for microtubules we have examined the relationship between Lewy bodies and mitochondrial loss. We have shown microtubule regression and mitochondrial and nuclear degradation in neurons with developing Lewy bodies. In PD, multiple Lewy bodies were often observed with α-synuclein interacting with DNA to cause marked nuclear degradation. In DLB, the mitochondria are drawn into the Lewy body and the mitochondrial integrity is lost. This work suggests that Lewy bodies are cytotoxic. In DLB, we suggest that microtubule regression and mitochondrial loss results in decreased cellular energy and axonal transport that leads to cell death. In PD, α-synuclein aggregations are associated with intact mitochondria but interacts with and causes nuclear degradation which may be the major cause of cell death., (© 2015 International Society of Neuropathology.)

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well., Competing Interests: The authors declare no conflict of interest.

Unlabelled: Comparison with existing methods., Background: Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue., Method: Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions., Results: The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue. A 2D-DIGE comparison of purified GCIs (n=2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively., Conclusions: This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In spite of definite roles for β-amyloid (Aβ) in familial Alzheimer's disease (AD), the cause of sporadic AD remains unknown. Amyloid senile plaques and Lewy body pathology frequently coexist in neocortical and hippocampal regions of AD and Parkinson's diseases. However, the relationship between Aβ and α-synuclein (α-Syn), the principle components in the pathological structures, in neuronal toxicity and the mechanisms of their interaction are not well studied. As Aβ and α-Syn accumulate in aging patients, the biological functions and toxicity of these polypeptides in the aging brain may be different from those in young brain. We examined the neurotoxicity influences of Aβ1-42 or α-Syn on mature neurons and the effects of Aβ1-42 or α-Syn on the production of endogenous α-Syn or Aβ1-40 reciprocally using a model of culture enriched with primary neurons from the hippocampus of adult rats. Treatment of neurons with high concentrations of Aβ1-42 or α-Syn caused significant apoptosis of neurons. Following Aβ1-42 treatment at sub apoptotic concentrations, both intra- and extra-cellular α-Syn levels were significantly increased. Reciprocally, the non-toxic levels of α-Syn treatment also increased intra- and extra-cellular Aβ1-40 levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, suppressed α-Syn-induced Aβ1-40 elevation, as well as Aβ1-42-induced α-Syn elevation. Thus, high concentrations of Aβ1-42 and α-Syn exert toxic effects on mature neurons; however, non-toxic concentration treatment of these polypeptides induced the production of each other reciprocally with possible involvement of PI3K pathway.

Background: The A53T mutation in the α-synuclein gene causes autosomal-dominant Lewy body Parkinson's disease (PD). Cultured cell models have linked this mutation to increased cell macroautophagy, although evidence of enhanced macroautophagy in patients with this mutation has not been assessed., Objective: To determine whether macroautophagy is increased by the A53T α-synuclein gene mutation in PD patients and cell models., Methods: Formalin-fixed paraffin-embedded 10 μm-thick tissue sections from the substantia nigra and anterior cingulate cortex of two PD patients with the A53T α-synuclein gene mutation were compared with four sporadic PD cases and four controls obtained from the Sydney Brain Bank. Lewy bodies were isolated from frontal cortex of a case with late stage PD (recruited from South Australian Brain Bank). Immunohistochemistry was performed for α-synuclein and the macroautophagy markers autophagy-specific gene (ATG) 5, ATG6/Beclin1 and ATG8/LC3. SH-SY5Y cells were transfected with wild type or A53T mutant α-synuclein plasmids and observable changes in macroautophagy marker protein levels assessed using Western blotting., Results: α-Synuclein immunoreactive neurites and dots were more numerous in patients with A53T mutations compared with late stage sporadic PD patients, and perinuclear cytoplasmic α-synuclein aggregates were observed in the α-synuclein A53T gene transfected SH-SY5Y cells compared to wild type transfections. All PD patients (with or without A53T mutations) had increased immunohistochemical evidence for macroautophagy compared with controls, and the levels of the ATG5 complex were equally increased in wild type and A53T α-synuclein gene transfected cells compared to controls., Conclusion: Despite increased α-synuclein accumulation with A53T mutations, macroautophagy is not increased above that observed in sporadic patients with PD or in cells transfected with wild type α-synuclein, suggesting that mutated α-synuclein protein is not removed by macroautophagy.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of alpha-synuclein (alpha-syn) in Parkinson disease. Herein we demonstrate that alpha-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated alpha-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P alpha-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric alpha-syn. These studies demonstrated that phosphorylation at S87 expands the structure of alpha-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of alpha-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in alpha-syn normal biology.

Neuronal intranuclear inclusion disease, a progressive ataxia that may be familial or sporadic, is characterized by numerous neuronal intranuclear inclusion bodies similar to those found in polyglutamine repeat diseases. Previously, we found that the intranuclear inclusion bodies are intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins in a similar way to ubiquitin. To identify the SUMO-1-associated proteins in the inclusion bodies, we isolated intranuclear inclusion bodies from fresh, frozen brain tissue of a case with familial neuronal intranuclear inclusion disease and solubilized the proteins. SUMO-1-associated inclusion body proteins were then immunocaptured using an anti-SUMO-1 antibody. The proteins, NSF, dynamin-1 and Unc-18-1 (rbSEC1), involved in membrane trafficking of proteins, and the chaperone HSP90, were identified following anti-SUMO-1-immunocapture by using tandem mass spectrometry and database searching. Immunohistochemistry of brain sections and crude brain homogenates of three cases of familial neuronal intranuclear inclusion disease confirmed the presence of these proteins in intranuclear inclusions.

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient's with Alzheimer's disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a "halo" around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to A beta peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

Ubiquitin is one of the major components of Lewy bodies (LB), the pathological hallmark of Parkinson's disease (PD). Here, we identified that a phosphorylated form of IkappaBalpha (pIkappaBalpha), an inhibitor of NF-kappaB, and SCF(beta-TrCP), the ubiquitin ligase of pIkappaBalpha, are components of LB in brains of PD patients. In vitro studies identified those proteins in the ubiquitin- and alpha-synuclein (known as the major component of LB)-positive LB-like inclusions generated in dopaminergic SH-SY5Y cells treated with MG132, a proteasome inhibitor. Intriguingly, IkappaBalpha migration into such ubiquitinated inclusions in cells treated with MG132 was inhibited by a cell-permeable peptide known to block phosphorylation of IkappaBalpha, although this peptide did not influence cell viability under proteasomal inhibition. Our results indicate that phosphorylation of IkappaBalpha plays a role in the formation of IkappaBalpha-containing inclusions caused by proteasomal dysfunction, and that the generation of such inclusion is independent of cell death caused by impairment of proteasome.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated.