[COVID-19 and the central and peripheral nervous system]. / COVID-19: Auswirkungen auf das zentrale und periphere Nervensystem.

The health effects of coronavirus disease 2019 (COVID-19) caused by the infection of SARS-CoV­2 (severe acute respiratory syndrome coronavirus 2) are becoming increasingly clear as the pandemic spreads. In addition to the lungs, other organs are also affected, which can significantly influence morbidity and mortality. In particular, neurological symptoms involving the central nervous system can lead to acute or long-term consequences. The mechanisms of this neuropathogenesis of SARS-CoV­2 infection and its relation to acute and chronic neurological symptoms are the subject of current studies investigating a potential direct and indirect viral infection of the nervous system. The following review summarizes the current status of neuropathological manifestations, molecular pathogenesis, possible infection pathways in the nervous system, and systemic effects. In addition, an overview of the Germany-wide CNS-COVID19 registry and collaborations is presented, which should contribute to a better understanding of the neurological symptoms of COVID-19.

Dysregulated autophagy in restrictive cardiomyopathy due to Pro209Leu mutation in BAG3.

Myofibrillary myopathies (MFM) are hereditary myopathies histologically characterized by degeneration of myofibrils and aggregation of proteins in striated muscle. Cardiomyopathy is common in MFM but the pathophysiological mechanisms are not well understood. The BAG3-Pro209Leu mutation is associated with early onset MFM and severe restrictive cardiomyopathy (RCM), often necessitating heart transplantation during childhood. We report on a young male patient with a BAG3-Pro209Leu mutation who underwent heart transplantation at eight years of age. Detailed morphological analyses of the explanted heart tissue showed intracytoplasmic inclusions, aggregation of BAG3 and desmin, disintegration of myofibers and Z-disk alterations. The presence of undegraded autophagosomes, seen by electron microscopy, as well as increased levels of p62, LC3-I and WIPI1, detected by immunohistochemistry and western blot analyses, indicated a dysregulation of autophagy. Parkin and PINK1, proteins involved in mitophagy, were slightly increased whereas mitochondrial OXPHOS activities were not altered. These findings indicate that altered autophagy plays a role in the pathogenesis and rapid progression of RCM in MFM caused by the BAG3-Pro209Leu mutation, which could have implications for future therapeutic strategies.

Application of CRISPR/Cas9 to Autophagy Research.

The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.

EphrinB2 repression through ZEB2 mediates tumour invasion and anti-angiogenic resistance.

Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies.

Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways.

Signal transmission by the noncanonical IkappaB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKKɛ, requires interaction with adapter proteins such as TRAF associated NF-κB activator (TANK). Although increased expression or dysregulation of both kinases has been described for a variety of human cancers, this study shows that deregulated expression of the TANK protein is frequently occurring in glioblastomas (GBMs). The functional relevance of TANK was analyzed in a panel of GBM-derived cell lines and revealed that knockdown of TANK arrests cells in the S-phase and prohibits tumor cell migration. Deregulated TANK expression affects several signaling pathways controlling cell proliferation and the inflammatory response. Interference with stoichiometrically assembled signaling complexes by overexpression or silencing of TANK prevented constitutive interferon-regulatory factor 3 (IRF3) phosphorylation. Knockdown of TANK frequently prevents constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). TANK-mediated ERK1/2 activation is independent from the canonical MAP kinase or ERK kinase (MEK) 1/2-mediated pathway and utilizes an alternative pathway that uses a TBK1/IKKɛ/Akt signaling axis, thus identifying a novel pathway suitable to block constitutive ERK1/2 activity.

Spinocerebellar ataxia type 2 with glial cell cytoplasmic inclusions.

Glial cell cytoplasmic inclusions were identified in a case of spinocerebellar ataxia type 2. These have not been reported before. The inclusions were found in low frequency in the dentate nucleus, cerebellar white matter, pontine transverse fibres, and the inferior olivary nucleus. They were of variable size and shape and expressed ubiquitin, thus resembling glial cytoplasmic inclusions in multiple system atrophy. However, their immunohistochemical profile was different as they did not show immunoreactivity for either tau protein or alpha-synuclein. There was no evidence of expanded polyglutamine tracts in these inclusions, which also failed to label with silver stains. As in many other neurodegenerative diseases, in spinocerebellar ataxia type 2 there may be pathogenic contributions of glial cells other than the common astrogliotic response to neuronal damage.

Cell type specific expression of vascular endothelial growth factor and angiopoietin-1 and -2 suggests an important role of astrocytes in cerebellar vascularization.

The vascularization of the central nervous system occurs by angiogenic sprouting, a process in which different factors like vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1/2 must act in a coordinated fashion. We investigated how these factors participate in capillarization of the cerebellum, an area experiencing marked reorganization processes during its postnatal development. VEGF and Ang-1 mRNA were predominantly expressed by astrocytes, while Ang-2 mRNA was specifically induced at the tip of invading endothelial cell cords. Similar to the cerebral cortex, vascularization of the cerebellum occurred in an inside-out pattern, following closely the generation and differentiation of each cerebellar layer. VEGF and Ang-1/2 expression patterns shifted in a similar inside-out fashion, supporting their proposed function in vessel growth and maturation.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.

Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants.

Tumor growth is angiogenesis-dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia-mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 are up-regulated in the tumor endothelium. A second endothelial cell-specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin-1 and the inhibitory ligand angiopoietin-2, has been characterized. We have examined 6 human primary breast-cancer samples and 4 murine breast-cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin-1, angiopoietin-2 and tie2 was compared to VEGF and VEGFR-2 expression. Human tumors expressed VEGFR-2 and tie2 but varied considerably in VEGF and angiopoietin-1/-2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR-2 and angiopoietin-2 but not tie2 or angiopoietin-1, suggesting activation of VEGFR-2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR-2, tie2 and angiopoietin-1 but little angiopoietin-2, suggesting activation of both VEGFR-2 and tie2 signaling pathways. In vivo studies using truncated dominant-negative tie2 and VEGFR-2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR-2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR-2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR-2 but suggest that, in addition, tie2-dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.

Is there a functioning vestibulocochlear nerve? Cochlear implantation in a child with symmetrical auditory findings but asymmetric imaging.

The finding of an abnormally narrow internal auditory meatus during the assessment of a child for cochlear implantation raises the possibility that the meatus may not contain the normal number of nerves. Even with currently available MRI techniques it may be extremely difficult to decide whether or not to offer cochlear implant in such cases. We present a child of 4 1/2 years, assessed for cochlear implantation. MRI and CT imaging suggested aplasia of one vestibulocochlear nerve and hypoplasia of the other. However, audiological tests showed clear responses to sound in both ears and functional use of sound in her daily life. This child was eventually implanted with encouraging postoperative results.

Up-regulation of hypoxia-inducible factors HIF-1alpha and HIF-2alpha under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function.

Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix-loop-helix PAS family, composed of alpha and beta subunits. HIF-1alpha shares 48 per cent identity with the recently identified HIF-2alpha protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von Hippel-Lindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2alpha mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in a range of VHL wildtype and VHL deficient RCC cell lines. In the presence of functional VHL protein, HIF-1alpha mRNA levels are elevated, whereas HIF-2alpha mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL deficient cell lines, both HIF-alpha subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1alpha and HIF-2alpha proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1alpha and HIF-2alpha in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1alpha and HIF-2alpha proteins.

Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat.

Vascular endothelial growth factor (VEGF), a key regulator of vasculogenesis and embryonic angiogenesis, was recently found to be up-regulated in an animal model of stroke. Unlike VEGF, angiopoietin (Ang)-1 and -2, their receptor tie-2, and the associated receptor tie-1 exert their functions at later stages of vascular development, i.e., during vascular remodeling and maturation. To assess the role of the angiopoietin/tie family in ischemia-triggered angiogenesis we analyzed their temporal and spatial expression pattern after middle cerebral artery occlusion (MCAO) using in situ hybridization and immunohistochemistry. Ang-1 mRNA was constitutively expressed in a subset of glial and neuronal cells with no apparent change in expression after MCAO. Ang-2 mRNA was up-regulated 6 hours after MCAO and was mainly observed in endothelial cell (EC) cord tips in the peri-infarct and infarct area. Up-regulation of both Ang-2 and VEGF coincided with EC proliferation. Interestingly, EC proliferation was preceded by a transient period of EC apoptosis, correlating with a change in VEGF/Ang-2 balance. Our observation of specific stages of vascular regression and growth after MCAO are in agreement with recent findings suggesting a dual role of Ang-2 in blood vessel formation, depending on the availability of VEGF.

Energy expenditure at rest and during standardized activities: a comparison between elderly and middle-aged women.

To estimate energy expenditure (EE) in elderly subjects, more age-specific data are required on energy costs of standardized activities. EE was assessed by using indirect calorimetry in 28 women aged 72 +/- 4 y (mean +/- SD) and in 29 middle-aged women (42 +/- 1 y) at rest (resting metabolic rate; RMR) and during sitting, sitting with standardized arm activity, and walking on a treadmill at 3 km/h. RMR and EE during sitting, and sitting with standardized arm activity did not differ significantly between the groups, although EE expressed as a ratio of arm activity to RMR (physical activity ratio, PAR) tended to be higher in the elderly subjects. Walking EE was significantly higher in the elderly women (16.4 +/- 4.0 kJ/min) than in the middle-aged women (12.7 +/- 2.3 kJ/min), also when expressed as PAR. It is suggested that elderly women walk less efficiently. Because PARs are frequently used to estimate daily EE, it is important to note that additional age-specific data might be required.

A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis.

Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 microM [35S]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37 degrees C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Q10 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [35S]L-cysteine: ala, ser, pro, val, gly, homocysteine, D- or L-penicillamine, arg, asp, or leu. Similarly, D-cysteine and beta-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and alpha-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an alpha-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteine-specific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.