Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase.

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. Here, we report a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. We establish the mitochondrial motor-adaptor protein Milton as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that the GlcNAcylation state of Milton is altered by extracellular glucose and that OGT alters mitochondrial motility in vivo. Our findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.

PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility.

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Molecular basis of F-actin regulation and sarcomere assembly via myotilin.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.

The mechanism of Ca2+ -dependent regulation of kinesin-mediated mitochondrial motility.

Mitochondria are mobile organelles and cells regulate mitochondrial movement in order to meet the changing energy needs of each cellular region. Ca(2+) signaling, which halts both anterograde and retrograde mitochondrial motion, serves as one regulatory input. Anterograde mitochondrial movement is generated by kinesin-1, which interacts with the mitochondrial protein Miro through an adaptor protein, milton. We show that kinesin is present on all axonal mitochondria, including those that are stationary or moving retrograde. We also show that the EF-hand motifs of Miro mediate Ca(2+)-dependent arrest of mitochondria and elucidate the regulatory mechanism. Rather than dissociating kinesin-1 from mitochondria, Ca(2+)-binding permits Miro to interact directly with the motor domain of kinesin-1, preventing motor/microtubule interactions. Thus, kinesin-1 switches from an active state in which it is bound to Miro only via milton, to an inactive state in which direct binding to Miro prevents its interaction with microtubules. Disrupting Ca(2+)-dependent regulation diminishes neuronal resistance to excitotoxicity.

Non-uniform sampling of similar NMR spectra and its application to studies of the interaction between alpha-synuclein and liposomes.

The accelerated acquisition of multidimensional NMR spectra using sparse non-uniform sampling (NUS) has been widely adopted in recent years. The key concept in NUS is that a major part of the data is omitted during measurement, and then reconstructed using, for example, compressed sensing (CS) methods. CS requires spectra to be compressible, that is, they should contain relatively few "significant" points. The more compressible the spectrum, the fewer experimental NUS points needed in order for it to be accurately reconstructed. In this paper we show that the CS processing of similar spectra can be enhanced by reconstructing only the differences between them. Accurate reconstruction can be obtained at lower sampling levels as the difference is sparser than the spectrum itself. In many situations this method is superior to "conventional" compressed sensing. We exemplify the concept of "difference CS" with one such case-the study of alpha-synuclein binding to liposomes and its dependence on temperature. To obtain information on temperature-dependent transitions between different states, we need to acquire several dozen spectra at various temperatures, with and without the presence of liposomes. Our detailed investigation reveals that changes in the binding modes of the alpha-synuclein ensemble are not only temperature-dependent but also show non-linear behavior in their transitions. Our proposed CS processing approach dramatically reduces the number of NUS points required and thus significantly shortens the experimental time.

APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer's pathology in trisomy 21 neurons.

Trisomy 21 (T21) causes Down syndrome and an early-onset form of Alzheimer's disease (AD). Here, we used human induced pluripotent stem cells (hiPSCs) along with CRISPR-Cas9 gene editing to investigate the contribution of chromosome 21 candidate genes to AD-relevant neuronal phenotypes. We utilized a direct neuronal differentiation protocol to bypass neurodevelopmental cell fate phenotypes caused by T21 followed by unbiased proteomics and western blotting to define the proteins dysregulated in T21 postmitotic neurons. We show that normalization of copy number of APP and DYRK1A each rescue elevated tau phosphorylation in T21 neurons, while reductions of RCAN1 and SYNJ1 do not. To determine the T21 alterations relevant to early-onset AD, we identified common pathways altered in familial Alzheimer's disease neurons and determined which of these were rescued by normalization of APP and DYRK1A copy number in T21 neurons. These studies identified disruptions in T21 neurons in both the axonal cytoskeletal network and presynaptic proteins that play critical roles in axonal transport and synaptic vesicle cycling. These alterations in the proteomic profiles have functional consequences: fAD and T21 neurons exhibit dysregulated axonal trafficking and T21 neurons display enhanced synaptic vesicle release. Taken together, our findings provide insights into the initial molecular alterations within neurons that ultimately lead to synaptic loss and axonal degeneration in Down syndrome and early-onset AD.

Rheumatoid neutrophilic dermatosis under treatment with the interleukin-6-receptor-antagonist sarilumab in a patient with seropositive rheumatoid arthritis.

Skin manifestations may arise as adverse events following the use of novel drugs. We report a case of a patient with seropositive rheumatoid arthritis who developed a rheumatoid neutrophilic dermatosis (RND) under treatment with the interleukin-6-receptor-antagonist sarilumab. The skin lesions developed 2-3 days after the first injection. RND presents with asymptomatic, symmetrical fixed urticarial-like papules, plaques, and nodules, localized typically on the extensor surfaces of the forearms and hands. After discontinuing the medication, the nodules in our patient disappeared within a month.

Pharmacokinetics and mass balance of vericiguat in rats and dogs and distribution in rats.

Vericiguat is a soluble guanylate cyclase stimulator. The pharmacokinetics, absorption, metabolism, and excretion properties of vericiguat in rats and dogs and the distribution in rats are reported. [14C]-labelled vericiguat was studied in intact and bile duct-cannulated rats (oral and intravenous administration), and dogs (oral administration).Vericiguat reached maximum plasma concentrations at 1-3 h after oral administration. Absolute bioavailability was moderate in rats and high in dogs. Vericiguat was the most abundant component in plasma of rats and dogs.After oral administration to rats, radioactivity was widely distributed. Penetration into the brain was minimal. Elimination was rapid from most tissues in rats. Most of the radioactivity was excreted in faeces (rat: 81%, dog: 89%), while low amounts were excreted in urine (rat: 11%, dog: 4%). Clearance routes in both species were unchanged excretion and metabolism via glucuronidation and oxidative reactions. After intravenous administration to bile duct-cannulated rats, a relevant proportion of the dose (30%) underwent direct excretion into the gastrointestinal tract as unchanged vericiguat.

Controllers of cutaneous regulatory T cells: ultraviolet radiation and the skin microbiome.

For the maintenance of homeostasis termination of immune reactions is as equally important as their induction. In this scenario regulatory T cells (Treg) play an important role. Accordingly a variety of inflammatory diseases are caused by an impairment of Treg. Hence, it is important to identify triggers by which Treg can be induced and activated, respectively. For quite a long time it is known that ultraviolet radiation can induce Treg which inhibit cutaneous immune reactions including contact hypersensitivity. Since these Treg inhibit in an antigen-specific fashion they may harbor therapeutic potential. However similar Treg can be induced also by other triggers which include vitamin D and antimicrobial peptides. Recently it was discovered that the gut microbiome controls the development of Treg in the intestine. The same may apply for the skin. Short chain fatty acids, microbiota-derived bacterial fermentation products, appear to induce and to activate Treg in the skin. Topical application of short chain fatty acids was shown to inhibit contact hypersensitivity and to reduce inflammation in the murine imiquimod-induced psoriasis-like skin inflammation model. Together, these data indicate that induction and activation of Treg may be a potential therapeutic strategy to treat inflammatory diseases in the future.

[Arthroderma crocatum on human skin]. / Arthroderma crocatum auf menschlicher Haut.

We report on three cases in which Arthroderma (A.) crocatum was isolated from human skin in Germany. The characteristics and epidemiology of this rare geophilic and probably mostly apathogenic dermatophyte are described paying special attention to its gymnothecia. The combination of KOH mount, culture and genetic analysis is the foundation for clinically meaningful conclusions. It is likely that the prevalence of A. crocatum is currently underestimated.

"Indian" strains of Trichophyton mentagrophytes with reduced itraconazole susceptibility in Germany.

We use published reports and three of our own tinea cases as an opportunity to report on "Indian" strains of Trichophyton (T.) mentagrophytes with ITS genotype VIII and reduced susceptibility to itraconazole due to the mutation c.1342G>A in the SQLE gene in Germany. In vitro measurements of resistance revealed normal susceptibility to terbinafine, but markedly reduced susceptibility to itraconazole - although no valid breakpoints are currently defined and minimum inhibitory concentrations (MICs) depend on the methods used. Problems related to the determination and interpretation of MICs are outlined. Our cases show that azole-resistant "Indian" strains of T. mentagrophytes with ITS genotype VIII occurred in Germany as early as 2011, which is earlier than was previously assumed. This variant of the pathogen cannot be phenotypically distinguished from customary strains of T. mentagrophytes; its identification is based on genetics. The taxonomic classification is still under debate. This variant is anthropophilic and causes only mildly inflammatory tinea lesions with many fungal elements. Its further dissemination must therefore be expected. Prerequisites for rapid and valid antimycotic testing against dermatophytes need to be developed.

From the (skin) doctor's office to the lecture hall: An innovative, practice-oriented, media-supported teaching project with supra-regional interdisciplinary usage options.

BACKGROUND: The transfer of practical knowledge and skills is the focus of modern medical teaching (master plan medical studies 2020). The aim of the teaching project is to provide medical students with important dermatological learning goals and contents by using innovative methods. METHODS: As part of a teaching project funded by the Federal Ministry of Education and Research (BMBF) at the Department of Dermatology, University Hospital Schleswig-Holstein in Kiel, various new, partly media-supported teaching modules were developed in addition to curricular teaching and optimized by regular acceptance evaluations during the development process. RESULTS: (1.) Professionally created instructional movies present essential techniques for diagnosis and therapy: standardized dermatological whole-body examination, wound smear sampling, performing of biopsies, curettages and excisions as well as skin suturing techniques on exercise material and on patients. (2.) Tutor-based courses offer students the ability to practice these techniques independently. (3.) Seminar lectures show different clinical pictures in university medicine and doctor's offices as well as the important interaction between clinic and doctor's practice in patient care. (4.) One-day internships in a teaching practice convey the activity in this setting. (5.) Seminars on psychodermatology provide insight into the stress caused by the skin disease using the "bio-psychosocial disease model". So far, 282 students have participated in the modules. In 88-100 % of the evaluations, there was a desire for further expansion of the new courses and integration into curricular teaching. CONCLUSIONS: Our innovative teaching modules resulted in great acceptance by the students. The freely available instructional films were successfully used by other university locations due to networking in the Academic Teaching Forum. One perspective is the supra-regional and sustainable use of our teaching modules and the transfer of the concept to other departments and faculties.

A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities.

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of 'residual structure' or 'conformational preference'. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP [Formula: see text]-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.

Measurements of sun sensitivity in five European countries confirm the relative nature of Fitzpatrick skin phototype scale.

BACKGROUND/PURPOSE: Skin colour and sun sensitivity are highly related to the distance to the equator: people in southern latitudes are usually darker and less sensitive to sun than in northern latitudes. Whether differences in sun sensitivity can be found in a relatively homogenous European population is unclear. We aimed to objectively measure sun sensitivity (assessed as pigment protection factor (PPF)) in five European countries, relate it to self-assessed Fitzpatrick skin phototype (FST) and to determine whether PPF levels in the different FST categories are dependent on the investigated countries. METHODS: Volunteers (n = 569) were recruited in Copenhagen (Denmark), Dublin (Ireland), London (England), Münster (Germany) and Ioannina (Greece). Skin phototype was self-assessed using the FST scale. PPF was measured at both sun-protected buttocks and five sun-exposed skin sites by a skin reflectance spectrophotometer. RESULTS: Overall, there were statistically significant differences in PPF of the buttocks, inner arm, outer arm, forehead, chest and back between the five countries (P ≤ .031). Generally, PPF level was lower in northern than in southern latitudes. PPF of the buttocks was similar in all countries for those who identified as FST I (P = .723). However, it was statistically significantly different (P ≤ 2.913*10-4 ) and country-dependent for those who identified as FST II-IV. CONCLUSION: Objectively measured sun sensitivity is higher (lower PPF) in northern compared with southern latitudes. The choice of self-identified FST category is influenced by a person's immediate environment. Therefore, we confirmed the relative nature of the FST scale and the need to standardise the skin phototype assessment procedure.

The EANM practical guidelines for sentinel lymph node localisation in oral cavity squamous cell carcinoma.

PURPOSE: Sentinel lymph node biopsy is an essential staging tool in patients with clinically localized oral cavity squamous cell carcinoma. The harvesting of a sentinel lymph node entails a sequence of procedures with participation of specialists in nuclear medicine, radiology, surgery, and pathology. The aim of this document is to provide guidelines for nuclear medicine physicians performing lymphoscintigraphy for sentinel lymph node detection in patients with early N0 oral cavity squamous cell carcinoma. METHODS: These practice guidelines were written and have been approved by the European Association of Nuclear Medicine (EANM) and the International Atomic Energy Agency (IAEA) to promote high-quality lymphoscintigraphy. The final result has been discussed by distinguished experts from the EANM Oncology Committee, and national nuclear medicine societies. The document has been endorsed by the Society of Nuclear Medicine and Molecular Imaging (SNMMI). These guidelines, together with another two focused on Surgery and Pathology (and published in specialised journals), are part of the synergistic efforts developed in preparation for the "2018 Sentinel Node Biopsy in Head and Neck Consensus Conference". CONCLUSION: The present practice guidelines will help nuclear medicine practitioners play their essential role in providing high-quality lymphatic mapping for the care of early N0 oral cavity squamous cell carcinoma patients.

Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility.

The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.

O-GlcNAc Transferase Is Essential for Sensory Neuron Survival and Maintenance.

O-GlcNAc transferase (OGT) regulates a wide range of cellular processes through the addition of the O-GlcNAc sugar moiety to thousands of protein substrates. Because nutrient availability affects the activity of OGT, its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, but little is known about its importance in basic neuronal processes in vivo Here, we show that OGT is essential for sensory neuron survival and maintenance in mice. Sensory neuron-specific knock-out of OGT results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell-body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as animals age. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. The effects on neuronal health in vivo are not solely due to disruption of developmental processes, because inducing OGT knock-out in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve-ending loss occurs before a decrease in cell bodies; this phenotype is indicative of axonal dieback that progresses to neuronal death. Our findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons.SIGNIFICANCE STATEMENT We show the importance of O-GlcNAc transferase (OGT) for sensory neuron health and survival in vivo This study is the first to find that loss of OGT results in neuronal cell death. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neuropathy. The sensory neurons lie outside of the blood-brain barrier and therefore, compared to central neurons, may have a greater need for mechanisms of metabolic sensing and compensation. Peripheral sensory neurons in particular are subject to degeneration in diabetes. Our findings provide a foundation for understanding the role of OGT under normal physiological conditions in the peripheral nervous system. This knowledge will be important for gaining greater insight into such disease states as diabetic neuropathy.

Biotransformation of Finerenone, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, in Dogs, Rats, and Humans, In Vivo and In Vitro.

Mass balance and biotransformation of finerenone, a nonsteroidal mineralocorticoid receptor antagonist, were investigated in four healthy male volunteers following a single oral administration of 10 mg (78 µCi) of [14C]finerenone and compared with data from studies in dogs and rats. The total recovery of the administered radioactivity was 101% in humans, 94.7% in dogs, and 95.2% in rats. In humans, radioactivity was mainly excreted renally (80%); in rats, it was primarily the biliary/fecal route (76%); and in dogs, excretion was more balanced. Finerenone was extensively metabolized in all species by oxidative biotransformation, with minor amounts of unchanged drug in excreta (humans: 1%; dogs, rats: <9%). In vitro studies suggested cytochrome P450 3A4 was the predominant enzyme involved in finerenone metabolism in humans. Primary metabolic transformation involved aromatization of the dihydronaphthyridine moiety of metabolite M1 as a major clearance pathway with a second oxidative pathway leading to M4. These were both prone to further oxidative biotransformation reactions. Naphthyridine metabolites (M1-M3) were the dominant metabolites identified in human plasma, with no on-target pharmacological activity. In dog plasma, finerenone and metabolite M2 constituted the major components; finerenone accounted almost exclusively for drug-related material in rat plasma. For metabolites M1-M3, axial chirality was observed, represented by two atropisomers (e.g., M1a and M1b). Analysis of plasma and excreta showed one atropisomer (a-series, >79%) of each metabolite predominated in all three species. In summary, the present study demonstrates that finerenone is cleared by oxidative biotransformation, mainly via naphthyridine derivatives.