Clinical and radiological outcome following trauma-related reverse shoulder arthroplasty.

BACKGROUND: Reverse shoulder arthroplasty (RSA) is a frequently used therapy for complex proximal humeral fractures and posttraumatic disorders. The present study's purpose was to assess the clinical and radiological outcome of primary and secondary RSA, and to analyze the impact of refixation of the greater tuberosity (GT). PATIENTS AND METHODS: 28 patients with primary fracture RSA and 18 patients with RSA due to posttraumatic disorders were examined with a mean clinical follow-up of 2.5 ± 1.73 years. Operative details and radiographs were retrospectively reviewed. Additional analyses were performed for healed and non-healed GT in primary RSA. RESULTS: Patients with fracture RSA had higher Constant-Murley score (CMS) than secondary RSA without reaching significance (p = 0.104). No significant difference was present for the quality of life measured by the Short Form 36 (SF 36) and the range of motion. In primary RSA, 78.6% GT healed anatomically. Compared to non-healed GT, patients with healed GT had a significantly higher CMS (p = 0.011), external rotation (p = 0.026) and forward flexion (p = 0.083), whereas DASH score was lower without a significant difference (p = 0.268). SF 36 showed no significant difference. Patients with healed GT had a more neutral glenoid version (p = 0.009). CONCLUSION: Superior range of motion and clinical outcome scores were present for anatomically healed GT. Therefore, refixation of the tuberosities is recommended. Secondary RSA can result in inferior results compared to primary RSA, so patients need to be adequately informed.

Astrocytic uptake of posttranslationally modified amyloid-ß leads to endolysosomal system disruption and induction of pro-inflammatory signaling.

The disruption of astrocytic catabolic processes contributes to the impairment of amyloid-ß (Aß) clearance, neuroinflammatory signaling, and the loss of synaptic contacts in late-onset Alzheimer's disease (AD). While it is known that the posttranslational modifications of Aß have significant implications on biophysical properties of the peptides, their consequences for clearance impairment are not well understood. It was previously shown that N-terminally pyroglutamylated Aß3(pE)-42, a significant constituent of amyloid plaques, is efficiently taken up by astrocytes, leading to the release of pro-inflammatory cytokine tumor necrosis factor α and synapse loss. Here we report that Aß3(pE)-42, but not Aß1-42, gradually accumulates within the astrocytic endolysosomal system, disrupting this catabolic pathway and inducing the formation of heteromorphous vacuoles. This accumulation alters lysosomal kinetics, lysosome-dependent calcium signaling, and upregulates the lysosomal stress response. These changes correlate with the upregulation of glial fibrillary acidic protein (GFAP) and increased activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Treatment with a lysosomal protease inhibitor, E-64, rescues GFAP upregulation, NF-κB activation, and synapse loss, indicating that abnormal lysosomal protease activity is upstream of pro-inflammatory signaling and related synapse loss. Collectively, our data suggest that Aß3(pE)-42-induced disruption of the astrocytic endolysosomal system leads to cytoplasmic leakage of lysosomal proteases, promoting pro-inflammatory signaling and synapse loss, hallmarks of AD-pathology.

A2A adenosine receptor-driven cAMP signaling in olfactory bulb astrocytes is unaffected in experimental autoimmune encephalomyelitis.

Introduction: The cyclic nucleotide cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger, which is known to play an important anti-inflammatory role. Astrocytes in the central nervous system (CNS) can modulate inflammation but little is known about the significance of cAMP in their function. Methods: We investigated cAMP dynamics in mouse olfactory bulb astrocytes in brain slices prepared from healthy and experimental autoimmune encephalomyelitis (EAE) mice. Results: The purinergic receptor ligands adenosine and adenosine triphosphate (ATP) both induced transient increases in cAMP in astrocytes expressing the genetically encoded cAMP sensor Flamindo2. The A2A receptor antagonist ZM241385 inhibited the responses. Similar transient increases in astrocytic cAMP occurred when olfactory receptor neurons were stimulated electrically, resulting in ATP release from the stimulated axons that increased cAMP, again via A2A receptors. Notably, A2A-mediated responses to ATP and adenosine were not different in EAE mice as compared to healthy mice. Discussion: Our results indicate that ATP, synaptically released by afferent axons in the olfactory bulb, is degraded to adenosine that acts on A2A receptors in astrocytes, thereby increasing the cytosolic cAMP concentration. However, this pathway is not altered in the olfactory bulb of EAE mice.

Knockdown of Na,K-ATPase ß-subunits in Oncopeltus fasciatus induces molting problems and alterations in tracheal morphology.

The ubiquitously expressed transmembrane enzyme Na,K-ATPase (NKA) is vital in maintaining functionality of cells. The association of α- and ß-subunits is believed to be essential for forming a functional enzyme. In the large milkweed bug Oncopeltus fasciatus four α1-paralogs and four ß-subunits exist that can associate into NKA complexes. This diversity raises the question of possible tissue-specific distribution and function. While the α1-subunits are known to modulate cardenolide-resistance and ion-transport efficiency, the functional importance of the ß-subunits needed further investigation. We here characterize all four different ß-subunits at the cellular, tissue, and whole organismal scales. A knockdown of different ß-subunits heavily interferes with molting success resulting in strongly hampered phenotypes. The failure of ecdysis might be related to disrupted septate junction (SJ) formation, also reflected in ß2-suppression-induced alteration in tracheal morphology. Our data further suggest the existence of isolated ß-subunits forming homomeric or ß-heteromeric complexes. This possible standalone and structure-specific distribution of the ß-subunits predicts further, yet unknown pump-independent functions. The different effects caused by ß knockdowns highlight the importance of the various ß-subunits to fulfill tissue-specific requirements.

Role of P2Y receptors in astrocyte physiology and pathophysiology.

Astrocytes are active constituents of the brain that manage ion homeostasis and metabolic support of neurons and directly tune synaptic transmission and plasticity. Astrocytes express all known P2Y receptors. These regulate a multitude of physiological functions such as cell proliferation, Ca2+ signalling, gliotransmitter release and neurovascular coupling. In addition, P2Y receptors are fundamental in the transition of astrocytes into reactive astrocytes, as occurring in many brain disorders such as neurodegenerative diseases, neuroinflammation and epilepsy. This review summarizes the current literature addressing the function of P2Y receptors in astrocytes in the healthy brain as well as in brain diseases.

Three-dimensional analyses of vascular network morphology in a murine lymph node by X-ray phase-contrast tomography with a 2D Talbot array.

With growing molecular evidence for correlations between spatial arrangement of blood vasculature and fundamental immunological functions, carried out in distinct compartments of the subdivided lymph node, there is an urgent need for three-dimensional models that can link these aspects. We reconstructed such models at a 1.84 µm resolution by the means of X-ray phase-contrast imaging with a 2D Talbot array in a short time without any staining. In addition reconstructions are verified in immunohistochemistry staining as well as in ultrastructural analyses. While conventional illustrations of mammalian lymph nodes depict the hilus as a definite point of blood and lymphatic vessel entry and exit, our method revealed that multiple branches enter and emerge from an area that extends up to one third of the organ's surface. This could be a prerequisite for the drastic and location-dependent remodeling of vascularization, which is necessary for lymph node expansion during inflammation. Contrary to corrosion cast studies we identified B-cell follicles exhibiting a two times denser capillary network than the deep cortical units of the T-cell zone. In addition to our observation of high endothelial venules spatially surrounding the follicles, this suggests a direct connection between morphology and B-cell homing. Our findings will deepen the understanding of functional lymph node composition and lymphocyte migration on a fundamental basis.

Structural deficits in key domains of Shank2 lead to alterations in postsynaptic nanoclusters and to a neurodevelopmental disorder in humans.

Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(-2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans.

Young COVID-19 Patients Show a Higher Degree of Microglial Activation When Compared to Controls.

The severe acute respiratory syndrome-corona virus type 2 (SARS-CoV-2) is the cause of human coronavirus disease 2019 (COVID-19). Since its identification in late 2019 SARS-CoV-2 has spread rapidly around the world creating a global pandemic. Although considered mainly a respiratory disease, COVID-19 also encompasses a variety of neuropsychiatric symptoms. How infection with SARS-CoV-2 leads to brain damage has remained largely elusive so far. In particular, it has remained unclear, whether signs of immune cell and / or innate immune and reactive astrogliosis are due to direct effects of the virus or may be an expression of a non-specific reaction of the brain to a severe life-threatening disease with a considerable proportion of patients requiring intensive care and invasive ventilation activation. Therefore, we designed a case-control-study of ten patients who died of COVID-19 and ten age-matched non-COVID-19-controls to quantitatively assess microglial and astroglial response. To minimize possible effects of severe systemic inflammation and / or invasive therapeutic measures we included only patients without any clinical or pathomorphological indication of sepsis and who had not been subjected to invasive intensive care treatment. Our results show a significantly higher degree of microglia activation in younger COVID-19 patients, while the difference was less and not significant for older COVID-19 patients. The difference in the degree of reactive gliosis increased with age but was not influenced by COVID-19. These preliminary data warrants further investigation of larger patient cohorts using additional immunohistochemical markers for different microglial phenotypes.

A glibenclamide-sensitive TRPM4-mediated component of CA1 excitatory postsynaptic potentials appears in experimental autoimmune encephalomyelitis.

The transient receptor potential melastatin 4 (TRPM4) channel contributes to disease severity in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and to neuronal cell death in models of excitotoxicity and traumatic brain injury. As TRPM4 is activated by intracellular calcium and conducts monovalent cations, we hypothesized that TRPM4 may contribute to and boost excitatory synaptic transmission in CA1 pyramidal neurons of the hippocampus. Using single-spine calcium imaging and electrophysiology, we found no effect of the TRPM4 antagonists 9-phenanthrol and glibenclamide on synaptic transmission in hippocampal slices from healthy mice. In contrast, glibenclamide but not 9-phenanthrol reduced excitatory synaptic potentials in slices from EAE mice, an effect that was absent in slices from EAE mice lacking TRPM4. We conclude that TRPM4 plays little role in basal hippocampal synaptic transmission, but a glibenclamide-sensitive TRPM4-mediated contribution to excitatory postsynaptic responses is upregulated at the acute phase of EAE.

Na,K-ATPase α1 and ß-subunits show distinct localizations in the nervous tissue of the large milkweed bug.

The Na,K-ATPase (NKA) is an essential ion transporter and signaling molecule in all animal tissues and believed to consist at least one α and one ß-subunit to form a functional enzyme. In the large milkweed bug, Oncopeltus fasciatus, adaptation to dietary cardiac glycosides (CGs), which can fatally block the NKA, has resulted in gene duplications leading to four α1-subunits. These differ in sensitivity to CGs, but resistance trades off against ion pumping activity, thus influencing the α1-subunits' suitability for specific tissues. Besides, O. fasciatus possesses four different ß-subunits that can alter the NKA's kinetics and should play an essential role in the formation of cellular junctions.Proteomic analyses revealed the distribution and composition of α1/ß-complexes in the nervous tissue of O. fasciatus. The highly CG-resistant, but less active α1B and the highly active, but less resistant α1C predominated in the nervous tissue and co-occurred with ß2 and ß3, partly forming larger complexes than just heterodimers. Immunohistochemical analyses provided a fine scale resolution of the subunits' distribution in different morphological structures of the nervous tissue. This may suggest that α1 as well as ß-subunits occur in isolation without the other subunit, which contradicts the present understanding that the two types of subunits have to associate to form functional complexes. An isolated occurrence was especially prominent for ß3 and ßx, the enigmatic fourth and N-terminally largely truncated ß-subunit. We hypothesize that dimerization of these ß-subunits plays a role in cell-cell contacts.

Time-Dependent Image Restoration of Low-SNR Live-Cell Ca2 Fluorescence Microscopy Data.

Live-cell Ca2+ fluorescence microscopy is a cornerstone of cellular signaling analysis and imaging. The demand for high spatial and temporal imaging resolution is, however, intrinsically linked to a low signal-to-noise ratio (SNR) of the acquired spatio-temporal image data, which impedes on the subsequent image analysis. Advanced deconvolution and image restoration algorithms can partly mitigate the corresponding problems but are usually defined only for static images. Frame-by-frame application to spatio-temporal image data neglects inter-frame contextual relationships and temporal consistency of the imaged biological processes. Here, we propose a variational approach to time-dependent image restoration built on entropy-based regularization specifically suited to process low- and lowest-SNR fluorescence microscopy data. The advantage of the presented approach is demonstrated by means of four datasets: synthetic data for in-depth evaluation of the algorithm behavior; two datasets acquired for analysis of initial Ca2+ microdomains in T-cells; finally, to illustrate the transferability of the methodical concept to different applications, one dataset depicting spontaneous Ca2+ signaling in jGCaMP7b-expressing astrocytes. To foster re-use and reproducibility, the source code is made publicly available.

GFAP and desmin expression in lymphatic tissues leads to difficulties in distinguishing between glial and stromal cells.

Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.

Using Genetically Encoded Calcium Indicators to Study Astrocyte Physiology: A Field Guide.

Ca2+ imaging is the most frequently used technique to study glial cell physiology. While chemical Ca2+ indicators served to visualize and measure changes in glial cell cytosolic Ca2+ concentration for several decades, genetically encoded Ca2+ indicators (GECIs) have become state of the art in recent years. Great improvements have been made since the development of the first GECI and a large number of GECIs with different physical properties exist, rendering it difficult to select the optimal Ca2+ indicator. This review discusses some of the most frequently used GECIs and their suitability for glial cell research.

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors.

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

Norepinephrine-Induced Calcium Signaling and Store-Operated Calcium Entry in Olfactory Bulb Astrocytes.

It is well-established that astrocytes respond to norepinephrine with cytosolic calcium rises in various brain areas, such as hippocampus or neocortex. However, less is known about the effect of norepinephrine on olfactory bulb astrocytes. In the present study, we used confocal calcium imaging and immunohistochemistry in mouse brain slices of the olfactory bulb, a brain region with a dense innervation of noradrenergic fibers, to investigate the calcium signaling evoked by norepinephrine in astrocytes. Our results show that application of norepinephrine leads to a cytosolic calcium rise in astrocytes which is independent of neuronal activity and mainly mediated by PLC/IP3-dependent internal calcium release. In addition, store-operated calcium entry (SOCE) contributes to the late phase of the response. Antagonists of both α1- and α2-adrenergic receptors, but not ß-receptors, largely reduce the adrenergic calcium response, indicating that both α-receptor subtypes mediate norepinephrine-induced calcium transients in olfactory bulb astrocytes, whereas ß-receptors do not contribute to the calcium transients.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

Age-Dependent Heterogeneity of Murine Olfactory Bulb Astrocytes.

Astrocytes have a high impact on the structure of the central nervous system, as they control neural activity, development, and plasticity. Heterogeneity of astrocytes has been shown before, but so far only a few studies have demonstrated heterogeneous morphology of astrocytes concerning aging. In this study, we examined morphologic differences of astrocyte subpopulations in adult mice and the progression of these differences with age. We surveyed astrocytes in olfactory bulb slices of mice aged 3 months, 1 year and 2 years (three animals each age group), based on their appearance in anti-GFAP immunostaining. Based on this data we established three different types of astrocytes: type I (stellate), type II (elliptic), and type III (squid-like). We found that with the advanced age of the mice, astrocytes grow in size and complexity. Major changes occurred between the ages of 3 months and 1 year, while between 1 and 2 years no significant development in cell size and complexity could be detected. Our results show that astrocytes in the olfactory bulb are heterogeneous and undergo morphological transformation until late adolescence but not upon senescence. Structural plasticity is further substantiated by the expression of vimentin in some astrocyte processes in all age groups.

Dopamine-induced calcium signaling in olfactory bulb astrocytes.

It is well established that astrocytes respond to the major neurotransmitters glutamate and GABA with cytosolic calcium rises, whereas less is known about the effect of dopamine on astroglial cells. In the present study, we used confocal calcium imaging in mouse brain slices of the olfactory bulb, a brain region with a large population of dopaminergic neurons, to investigate calcium signaling evoked by dopamine in astrocytes. Our results show that application of dopamine leads to a dose-dependent cytosolic calcium rise in astrocytes (EC50 = 76 µM) which is independent of neuronal activity and mainly mediated by PLC/IP3-dependent internal calcium release. Antagonists of both D1- and D2-class dopamine receptors partly reduce the dopaminergic calcium response, indicating that both receptor classes contribute to dopamine-induced calcium transients in olfactory bulb astrocytes.