Burkhard Gess

Monocyte Chemoattractant Protein-1–Deficiency Results in Altered Blood–Brain Barrier Breakdown After Experimental Stroke

Background and Purpose— Stroke-induced blood–brain barrier (BBB)-disruption can contribute to further progression of cerebral damage. There is rising evidence for a strong involvement of chemokines in postischemic BBB-breakdown. In a previous study, we showed that monocyte chemoattractant protein-1 (MCP-1)–deficiency results in a markedly reduced inflammatory reaction with decreased levels of interleukin-6, interleukin-1&bgr;, and granulocyte colony-stimulating factor after experimental stroke. With MCP-1 as one of the key players in stroke-induced inflammation, in this study, we investigated the influence of MCP-1 on poststroke BBB-disruption as well as transcription/translation of BBB-related genes/proteins after cerebral ischemia. Methods— Sixteen wild-type and 16 MCP-1−/− mice were subjected to 30 minutes of middle cerebral artery occlusion. By injecting high molecular-tracer, we compared the degree of BBB-disruption after middle cerebral artery occlusion. Real-time polymerase chain reactions and Western blot technique were used to compare tight-junction gene expression, protein secretion, and BBB-leakage. Results— Here, we report that MCP-1–deficiency results in a reduced BBB-leakage and a diminished expression of BBB-related genes occludin, zonula occludens-1, and zonula occludens-2. Real-time polymerase chain reactions and Western blot analysis revealed elevated claudin-5–levels in MCP-1−/− animals. MCP-1–deficiency resulted in reduced infarct sizes and an increased vascular accumulation of fluorescein-isothiocyanate-albumin. Conclusions— The results of the study provide further insights into the molecular mechanisms of BBB-opening and may help to better understand the mechanisms of infarct development after cerebral ischemia.

Sodium-dependent vitamin C transporter 2 deficiency causes hypomyelination and extracellular matrix defects in the peripheral nervous system.

Ascorbic acid (vitamin C) is necessary for myelination of Schwann cell/neuron cocultures and has shown beneficial effects in the treatment of a Charcot-Marie-Tooth neuropathy 1A (CMT1A) mouse model. Although clinical studies revealed that ascorbic acid treatment had no impact on CMT1A, it is assumed to have an important function in peripheral nerve myelination and possibly in remyelination. However, the transport pathway of ascorbic acid into peripheral nerves and the mechanism of ascorbic acid function in peripheral nerves in vivo remained unclear. In this study, we used sodium-dependent vitamin C transporter 2-heterozygous (SVCT2+/−) mice to elucidate the functions of SVCT2 and ascorbic acid in the murine peripheral nervous system. SVCT2 and ascorbic acid levels were reduced in SVCT2+/− peripheral nerves. Morphometry of sciatic nerve fibers revealed a decrease in myelin thickness and an increase in G-ratios in SVCT2+/− mice. Nerve conduction velocities and sensorimotor performance in functional tests were reduced in SVCT2+/− mice. To investigate the mechanism of ascorbic acid function, we studied the expression of collagens in the extracellular matrix of peripheral nerves. Here, we show that expression of various collagen types was reduced in sciatic nerves of SVCT2+/− mice. We found that collagen gene transcription was reduced in SVCT2+/− mice but hydroxyproline levels were not, indicating that collagen formation was regulated on the transcriptional and not the posttranslational level. These results help to clarify the transport pathway and mechanism of action of ascorbic acid in the peripheral nervous system and may lead to novel therapeutic approaches to peripheral neuropathies by manipulation of SVCT2 function.

Sodium-Dependent Vitamin C Transporter 2 (SVCT2) Expression and Activity in Brain Capillary Endothelial Cells after Transient Ischemia in Mice

Expression and transport activity of Sodium-dependent Vitamin C Transporter 2 (SVCT2) was shown in various tissues and organs. Vitamin C was shown to be cerebroprotective in several animal models of stroke. Data on expression, localization and transport activity of SVCT2 after cerebral ischemia, however, has been scarce so far. Thus, we studied the expression of SVCT2 after middle cerebral artery occlusion (MCAO) in mice by immunohistochemistry. We found an upregulation of SVCT2 after stroke. Co-stainings with Occludin, Von-Willebrand Factor and CD34 demonstrated localization of SVCT2 in brain capillary endothelial cells in the ischemic area after stroke. Time-course analyses of SVCT2 expression by immunohistochemistry and western blots showed upregulation in the subacute phase of 2–5 days. Radioactive uptake assays using 14C-labelled ascorbic acid showed a significant increase of ascorbic acid uptake into the brain after stroke. Taken together, these results provide evidence for the expression and transport activity of SVCT2 in brain capillary endothelial cells after transient ischemia in mice. These results may lead to the development of novel neuroprotective strategies in stroke therapy.

HSJ1-related hereditary neuropathies Novel mutations and extended clinical spectrum

Objectives: To determine the nature and frequency of HSJ1 mutations in patients with hereditary motor and hereditary motor and sensory neuropathies. Methods: Patients were screened for mutations by genome-wide or targeted linkage and homozygosity studies, whole-exome sequencing, and Sanger sequencing. RNA and protein studies of skin fibroblasts were used for functional characterization. Results: We describe 2 additional mutations in the HSJ1 gene in a cohort of 90 patients with autosomal recessive distal hereditary motor neuropathy (dHMN) and Charcot-Marie-Tooth disease type 2 (CMT2). One family with a dHMN phenotype showed the homozygous splice-site mutation c.229+1G>A, which leads to retention of intron 4 in the HSJ1 messenger RNA with a premature stop codon and loss of protein expression. Another family, presenting with a CMT2 phenotype, carried the homozygous missense mutation c.14A>G (p.Tyr5Cys). This mutation was classified as likely disease-related by several automatic algorithms for prediction of possible impact of an amino acid substitution on the structure and function of proteins. Both mutations cosegregated with autosomal recessive inheritance of the disease and were absent from the general population. Conclusions: Taken together, in our cohort of 90 probands, we confirm that HSJ1 mutations are a rare but detectable cause of autosomal recessive dHMN and CMT2. We provide clinical and functional information on an HSJ1 splice-site mutation and report the detailed phenotype of 2 patients with CMT2, broadening the phenotypic spectrum of HSJ1-related neuropathies.

Sleep disorders in Charcot-Marie-Tooth disease type 1

Introduction Obstructive sleep apnoea (OSA) and restless legs syndrome (RLS) have been reported in Charcot-Marie-Tooth disease (CMT) type 1A and axonal subtypes of CMT, respectively. The aim of this case–control study was to investigate both prevalence and severity of OSA, RLS and periodic limb movements in sleep (PLMS) in adult patients with genetically proven CMT1. Patients and methods 61 patients with CMT1 and 61 insomnic control subjects were matched for age, sex, and Body Mass Index. Neurological disability in patients with CMT was assessed using the Functional Disability Scale (FDS). RLS diagnosis was based on a screening questionnaire and structured clinical interviews. All participants underwent overnight polysomnography. Results OSA was present in 37.7% of patients with CMT1 and 4.9% of controls (p<0.0001). The mean Apnoea Hypoponea Index (AHI) was significantly higher in patients with CMT1 than in control individuals (9.1/h vs 1.2/h). RLS was present in 40.9% of patients with CMT1 and in 16.4% of controls (p<0.001). In the CMT1 group, OSA was significantly more common in men and RLS in women. The AHI correlated with both age and the FDS score, the latter being a significant independent predictor of OSA. PLMS were found in 41.0% of patients with CMT1, but were not correlated with measures of sleep quality. Conclusions In addition to known risk factors, CMT may predispose to OSA. RLS is highly prevalent not only in axonal subtypes of CMT but also in primarily demyelinating subforms of CMT. PLMS are common in CMT1, but do not significantly impair sleep quality.

Altered Dynamics in the Circadian Oscillation of Clock Genes in Dermal Fibroblasts of Patients Suffering from Idiopathic Hypersomnia

From single cell organisms to the most complex life forms, the 24-hour circadian rhythm is important for numerous aspects of physiology and behavior such as daily periodic fluctuations in body temperature and sleep-wake cycles. Influenced by environmental cues – mainly by light input -, the central pacemaker in the thalamic suprachiasmatic nuclei (SCN) controls and regulates the internal clock mechanisms which are present in peripheral tissues. In order to correlate modifications in the molecular mechanisms of circadian rhythm with the pathophysiology of idiopathic hypersomnia, this study aimed to investigate the dynamics of the expression of circadian clock genes in dermal fibroblasts of idiopathic hypersomniacs (IH) in comparison to those of healthy controls (HC). Ten clinically and polysomnographically proven IH patients were recruited from the department of sleep medicine of the University Hospital of Muenster. Clinical diagnosis was done by two consecutive polysomnographies (PSG) and Multiple Sleep Latency Test (MSLT). Fourteen clinical healthy volunteers served as control group. Dermal fibroblasts were obtained via punch biopsy and grown in cell culture. The expression of circadian clock genes was investigated by semiquantitative Reverse Transcriptase-PCR qRT-PCR analysis, confirming periodical oscillation of expression of the core circadian clock genes BMAL1, PER1/2 and CRY1/2. The amplitude of the rhythmically expressed BMAL1, PER1 and PER2 was significantly dampened in dermal fibroblasts of IH compared to HC over two circadian periods whereas the overall expression of only the key transcriptional factor BMAL1 was significantly reduced in IH. Our study suggests for the first time an aberrant dynamics in the circadian clock in IH. These findings may serve to better understand some clinical features of the pathophysiology in sleep – wake rhythms in IH.

Caveolin-1–mediated internalization of the vitamin C transporter SVCT2 in microglia triggers an inflammatory phenotype

Internalization and degradation of a vitamin C transporter trigger activation of microglia. Vitamin C prevents microglia activation Changes in the abundance of ascorbate, the reduced form of vitamin C, in the central nervous system (CNS) alter neuronal function and are associated with neurodevelopmental and neurodegenerative disorders. Activation of microglia, which occurs in response to tissue damage or pathogens, also contributes to neurodegenerative disease. Portugal et al. showed that the plasma membrane sodium–vitamin C cotransporter 2 (SVCT2) was required for microglia homeostasis in the CNS. Decreasing the amount of SVCT2 in the plasma membrane reduced vitamin C uptake and triggered activation of both primary rodent and human microglia. Treating microglia with ascorbate or preventing the internalization of SVCT2 blocked activation of microglia. These results demonstrate that ascorbate plays an essential role in microglial homeostasis and may prevent the microglial activation that contributes to neurodegenerative disease. Vitamin C is essential for the development and function of the central nervous system (CNS). The plasma membrane sodium–vitamin C cotransporter 2 (SVCT2) is the primary mediator of vitamin C uptake in neurons. SVCT2 specifically transports ascorbate, the reduced form of vitamin C, which acts as a reducing agent. We demonstrated that ascorbate uptake through SVCT2 was critical for the homeostasis of microglia, the resident myeloid cells of the CNS that are essential for proper functioning of the nervous tissue. We found that depletion of SVCT2 from the plasma membrane triggered a proinflammatory phenotype in microglia and resulted in microglia activation. Src-mediated phosphorylation of caveolin-1 on Tyr14 in microglia induced the internalization of SVCT2. Ascorbate treatment, SVCT2 overexpression, or blocking SVCT2 internalization prevented the activation of microglia. Overall, our work demonstrates the importance of the ascorbate transport system for microglial homeostasis and hints that dysregulation of ascorbate transport might play a role in neurological disorders.

Unravelling crucial biomechanical resilience of myelinated peripheral nerve fibres provided by the Schwann cell basal lamina and PMP22

There is an urgent need for the research of the close and enigmatic relationship between nerve biomechanics and the development of neuropathies. Here we present a research strategy based on the application atomic force and confocal microscopy for simultaneous nerve biomechanics and integrity investigations. Using wild-type and hereditary neuropathy mouse models, we reveal surprising mechanical protection of peripheral nerves. Myelinated peripheral wild-type fibres promptly and fully recover from acute enormous local mechanical compression while maintaining functional and structural integrity. The basal lamina which enwraps each myelinated fibre separately is identified as the major contributor to the striking fibres resilience and integrity. In contrast, neuropathic fibres lacking the peripheral myelin protein 22 (PMP22), which is closely connected with several hereditary human neuropathies, fail to recover from light compression. Interestingly, the structural arrangement of the basal lamina of Pmp22−/− fibres is significantly altered compared to wild-type fibres. In conclusion, the basal lamina and PMP22 act in concert to contribute to a resilience and integrity of peripheral nerves at the single fibre level. Our findings and the presented technology set the stage for a comprehensive research of the links between nerve biomechanics and neuropathies.

Varying survival of motoneurons and activation of distinct molecular mechanism in response to altered peripheral myelin protein 22 gene dosage.

Alteration in the expression level of peripheral myelin protein 22 (PMP22) is the most frequent cause for demyelinating neuropathies of Charcot‐Marie‐Tooth type. Here, we demonstrate a loss of motoneurons (MNs) in the spinal cords from transgenic mice over‐expressing Pmp22 (Pmp22tg) while mice lacking Pmp22 [Pmp22ko; knockout (ko)] exhibited normal MN numbers at the symptomatic age of 60 days. In order to describe the molecular changes in affected MNs, these cells were isolated from lumbar spinal cords by laser‐capture microdissection. Remarkably, the MNs of the Pmp22ko and Pmp22tg mice showed different expression profiles because of the altered Pmp22 expression. The changes in the expression profile of MNs from Pmp22ko mice resemble those described in MNs from mice after nerve injury and included genes that had been described in neuronal growth and regeneration like Gap43 and Sprr11a. The changes detected in the expression pattern of MNs from Pmp22tg mice exhibited fewer similarities to other expression patterns. The specific expression pattern in the MNs of the Pmp22ko mice might contribute to the better survival of the MNs. Our study also revealed induction of genes like brain‐expressed X‐linked 1 (Bex1) and desmoplakin (Dsp) that had recently been found up‐regulated in MNs of human amyotrophic lateral sclerosis patients.

Combining Growth Factor and Bone Marrow Cell Therapy Induces Bleeding and Alters Immune Response After Stroke in Mice

Background and Purpose— Bone marrow cell (BMC)–based therapies, either the transplantation of exogenous cells or stimulation of endogenous cells by growth factors like the granulocyte colony–stimulating factor (G-CSF), are considered a promising means of treating stroke. In contrast to large preclinical evidence, however, a recent clinical stroke trial on G-CSF was neutral. We, therefore, aimed to investigate possible synergistic effects of co-administration of G-CSF and BMCs after experimental stroke in mice to enhance the efficacy compared with single treatments. Methods— We used an animal model for experimental stroke as paradigm to study possible synergistic effects of co-administration of G-CSF and BMCs on the functional outcome and the pathophysiological mechanism. Results— G-CSF treatment alone led to an improved functional outcome, a reduced infarct volume, increased blood vessel stabilization, and decreased overall inflammation. Surprisingly, the combination of G-CSF and BMCs abrogated G-CSFs’ beneficial effects and resulted in increased hemorrhagic infarct transformation, altered blood–brain barrier, excessive astrogliosis, and altered immune cell polarization. These increased rates of infarct bleeding were mainly mediated by elevated matrix metalloproteinase-9–mediated blood–brain barrier breakdown in G-CSF- and BMCs-treated animals combined with an increased number of dilated and thus likely more fragile vessels in the subacute phase after cerebral ischemia. Conclusions— Our results provide new insights into both BMC-based therapies and immune cell biology and help to understand potential adverse and unexpected side effects.