Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.
- MeSH
- demyelinizační nemoci * patologie MeSH
- glykosfingolipidy metabolismus MeSH
- lidé MeSH
- molekuly buněčné adheze metabolismus MeSH
- myelinová pochva metabolismus MeSH
- neurotrofní faktory metabolismus MeSH
- sulfoglykosfingolipidy * MeSH
- transportní proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Promotion of myelin repair in the context of demyelinating diseases such as multiple sclerosis (MS) still represents a clinical unmet need, given that this disease is not only characterized by autoimmune activities but also by impaired regeneration processes. Hence, this relates to replacement of lost oligodendrocytes and myelin sheaths-the primary targets of autoimmune attacks. Endogenous remyelination is mainly mediated via activation and differentiation of resident oligodendroglial precursor cells (OPCs), whereas its efficiency remains limited and declines with disease progression and aging. Teriflunomide has been approved as a first-line treatment for relapsing remitting MS. Beyond its role in acting via inhibition of de novo pyrimidine synthesis leading to a cytostatic effect on proliferating lymphocyte subsets, this study aims to uncover its potential to foster myelin repair. METHODS: Within the cuprizone mediated de-/remyelination model teriflunomide dependent effects on oligodendroglial homeostasis and maturation, related to cellular processes important for myelin repair were analyzed in vivo. Teriflunomide administration was performed either as pulse or continuously and markers specific for oligodendroglial maturation and mitochondrial integrity were examined by means of gene expression and immunohistochemical analyses. In addition, axon myelination was determined using electron microscopy. RESULTS: Both pulse and constant teriflunomide treatment efficiently boosted myelin repair activities in this model, leading to accelerated generation of oligodendrocytes and restoration of myelin sheaths. Moreover, teriflunomide restored mitochondrial integrity within oligodendroglial cells. CONCLUSIONS: The link between de novo pyrimidine synthesis inhibition, oligodendroglial rescue, and maintenance of mitochondrial homeostasis appears as a key for successful myelin repair and hence for protection of axons from degeneration.
The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.
- MeSH
- axony metabolismus MeSH
- centrální nervový systém metabolismus MeSH
- lidé MeSH
- lipidové dvojvrstvy * metabolismus MeSH
- myelinová pochva metabolismus MeSH
- myelinový proteolipidový protein * metabolismus MeSH
- protein - isoformy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Recently developed therapeutic approaches for the treatment of Huntington's disease (HD) require preclinical testing in large animal models. The minipig is a suitable experimental animal because of its large gyrencephalic brain, body weight of 70-100 kg, long lifespan, and anatomical, physiological and metabolic resemblance to humans. The Libechov transgenic minipig model for HD (TgHD) has proven useful for proof of concept of developing new therapies. However, to evaluate the efficacy of different therapies on disease progression, a broader phenotypic characterization of the TgHD minipig is needed. In this study, we analyzed the brain tissues of TgHD minipigs at the age of 48 and 60-70 months, and compared them to wild-type animals. We were able to demonstrate not only an accumulation of different forms of mutant huntingtin (mHTT) in TgHD brain, but also pathological changes associated with cellular damage caused by mHTT. At 48 months, we detected pathological changes that included the demyelination of brain white matter, loss of function of striatal neurons in the putamen and activation of microglia. At 60-70 months, we found a clear marker of neurodegeneration: significant cell loss detected in the caudate nucleus, putamen and cortex. This was accompanied by clusters of structures accumulating in the neurites of some neurons, a sign of their degeneration that is also seen in Alzheimer's disease, and a significant activation of astrocytes. In summary, our data demonstrate age-dependent neuropathology with later onset of neurodegeneration in TgHD minipigs.
- MeSH
- bílá hmota patologie ultrastruktura MeSH
- biologické markery metabolismus MeSH
- degenerace nervu patologie MeSH
- geneticky modifikovaná zvířata MeSH
- genotyp MeSH
- hmotnostní úbytek MeSH
- Huntingtonova nemoc patologie MeSH
- index tělesné hmotnosti MeSH
- lidé MeSH
- miniaturní prasata MeSH
- modely nemocí na zvířatech MeSH
- motorické korové centrum patologie ultrastruktura MeSH
- myelinová pochva metabolismus MeSH
- nucleus caudatus patologie ultrastruktura MeSH
- prasata MeSH
- protein huntingtin metabolismus MeSH
- proteinové agregáty MeSH
- stárnutí patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Directing the organization of cells into a tissue with defined architectures is one use of biomaterials for regenerative medicine. To this end, hydrogels are widely investigated as they have mechanical properties similar to native soft tissues and can be formed in situ to conform to a defect. Herein, we describe the development of porous hydrogel tubes fabricated through a two-step polymerization process with an intermediate microsphere phase that provides macroscale porosity (66.5%) for cell infiltration. These tubes were investigated in a spinal cord injury model, with the tubes assembled to conform to the injury and to provide an orientation that guides axons through the injury. Implanted tubes had good apposition and were integrated with the host tissue due to cell infiltration, with a transient increase in immune cell infiltration at 1 week that resolved by 2 weeks post injury compared to a gelfoam control. The glial scar was significantly reduced relative to control, which enabled robust axon growth along the inner and outer surface of the tubes. Axon density within the hydrogel tubes (1744 axons/mm2) was significantly increased more than 3-fold compared to the control (456 axons/mm2), with approximately 30% of axons within the tube myelinated. Furthermore, implantation of hydrogel tubes enhanced functional recovery relative to control. This modular assembly of porous tubes to fill a defect and directionally orient tissue growth could be extended beyond spinal cord injury to other tissues, such as vascular or musculoskeletal tissue. STATEMENT OF SIGNIFICANCE: Tissue engineering approaches that mimic the native architecture of healthy tissue are needed following injury. Traditionally, pre-molded scaffolds have been implemented but require a priori knowledge of wound geometries. Conversely, hydrogels can conform to any injury, but do not guide bi-directional regeneration. In this work, we investigate the feasibility of a system of modular hydrogel tubes to promote bi-directional regeneration after spinal cord injury. This system allows for tubes to be cut to size during surgery and implanted one-by-one to fill any injury, while providing bi-directional guidance. Moreover, this system of tubes can be broadly applied to tissue engineering approaches that require a modular guidance system, such as repair to vascular or musculoskeletal tissues.
- MeSH
- axony účinky léků patologie MeSH
- hydrogely farmakologie MeSH
- jizva patologie MeSH
- lokomoce účinky léků MeSH
- maleimidy chemie MeSH
- mikrosféry MeSH
- myelinová pochva účinky léků metabolismus MeSH
- myši inbrední C57BL MeSH
- neuroglie patologie MeSH
- polyethylenglykoly chemie MeSH
- polymerizace MeSH
- poranění míchy patologie patofyziologie MeSH
- poréznost MeSH
- reagencia zkříženě vázaná chemie MeSH
- regenerace nervu účinky léků MeSH
- tkáňové podpůrné struktury chemie MeSH
- zadní končetina účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
Tuberous Sclerosis Complex (TSC) is a genetic hamartoma syndrome frequently associated with severe intractable epilepsy. In some TSC patients epilepsy surgery is a promising treatment option provided that the epileptogenic zone can be precisely delineated. TSC brain lesions (cortical tubers) contain dysmorphic neurons, brightly eosinophilic giant cells and white matter alterations in various proportions. However, a histological classification system has not been established for tubers. Therefore, the aim of this study was to define distinct histological patterns within tubers based on semi-automated histological quantification and to find clinically significant correlations. In total, we studied 28 cortical tubers and seven samples of perituberal cortex from 28 TSC patients who had undergone epilepsy surgery. We assessed mammalian target of rapamycin complex 1 (mTORC1) activation, the numbers of giant cells, dysmorphic neurons, neurons, and oligodendrocytes, and calcification, gliosis, angiogenesis, inflammation, and myelin content. Three distinct histological profiles emerged based on the proportion of calcifications, dysmorphic neurons and giant cells designated types A, B, and C. In the latter two types we were able to subsequently associate them with specific features on presurgical MRI. Therefore, these histopathological patterns provide consistent criteria for improved definition of the clinico-pathological features of cortical tubers identified by MRI and provide a basis for further exploration of the functional and molecular features of cortical tubers in TSC.
- MeSH
- dítě MeSH
- dospělí MeSH
- epilepsie komplikace metabolismus patologie chirurgie MeSH
- glióza komplikace patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mozková kůra patologie chirurgie MeSH
- multiproteinové komplexy analýza metabolismus MeSH
- myelinová pochva metabolismus patologie MeSH
- neurony patologie MeSH
- předškolní dítě MeSH
- TOR serin-threoninkinasy analýza metabolismus MeSH
- tuberózní skleróza komplikace metabolismus patologie chirurgie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: The aim of the study was to determine changes of biomarkers of nervous tissue degradation in experimental model of osmotic blood-brain barrier opening or water intoxication and to find whether they correspond to changes in well defined clinical entities. METHODS: In the cerebro-spinal fluid taken via the suboccipital puncture, myelin basic protein (MBP ng/ml), neuron-specific enolase (NSE ng/ml) and TAU-protein (Tau pg/ml) were determined by ELISA in 19 controls and 29 experimental rats several hours or one week after the experimental intervention. RESULTS: Significant difference between the control and experimental groups was revealed only for the concentration of myelin basic protein. After the BBB opening, its level dramatically increased within hours and dropped back to control values within one week. Water intoxication induced only dilutional hypoproteinorachia. No significant changes were found in NSE and levels of TAU-protein were not detectable. CONCLUSION: 1. Increased permeability of cytoplasmic membranes induced by water intoxication does not alter any of monitored CSF biomarkers. 2. Osmotic opening of the BBB in vivo experiment without the presence of other pathological conditions leads to a damage of myelin, without impairment of neurons or their axons.
- MeSH
- axony MeSH
- biologické markery MeSH
- encefalitogenní základní proteiny MeSH
- hematoencefalická bariéra * metabolismus MeSH
- intoxikace vodou * MeSH
- krysa rodu rattus MeSH
- myelinová pochva metabolismus patologie MeSH
- osmotický tlak MeSH
- proteiny tau MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A number of clinical neurological pathologies are associated with increased permeability of the blood brain barrier (BBB). Induced changes of the homeostatic mechanisms in the brain microenvironment lead among others to cellular changes in the CNS. The question was whether some of these changes can be induced by osmotic opening of BBB in an in vivo experiment and whether they can be detected in cerebrospinal fluid (CSF). CSF was taken via the suboccipital puncture from 10 healthy rats and six rats after the osmotic opening of the BBB. In all 16 animals, concentration of myelin basic protein (MBP ng/ml), Neuron-specific enolase (NSE ng/ml) and Tau-protein (Tau pg/ml) were determined in CSF by ELISA. Values in both groups were statistically evaluated. Significant difference between the control and experimental group was revealed only for the concentration of myelin basic protein (p<0.01). The presented results indicate that osmotic opening of the BBB in vivo experiment without the presence of other pathological conditions of the brain leads to a damage of myelin, without impairment of neurons or their axons.
- MeSH
- biologické markery MeSH
- encefalitogenní základní proteiny MeSH
- fosfopyruváthydratasa MeSH
- hematoencefalická bariéra účinky léků metabolismus patologie MeSH
- mannitol toxicita MeSH
- myelinová pochva účinky léků metabolismus patologie MeSH
- nervová vlákna myelinizovaná účinky léků metabolismus patologie MeSH
- osmotický tlak MeSH
- permeabilita MeSH
- potkani Wistar MeSH
- proteiny tau MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Our previous experiments revealed that water intoxication and osmotic BBB disruption in the rat allow penetration of high-molecular substances into the brain and that resulting changes in the internal environment of the CNS lead to pathological development, such as the loss of integrity of myelin. The aim of the present study was to determine whether the previously described phenomena are associated with increased water content in the brain. To answer the question following methods were used: a) water intoxication: intraperitoneal administration of distilled water, b) osmotic BBB disruption: application of mannitol (20 %) selectively into the internal carotid artery, c) brain wet weight was measured after decapitation, and subsequently (after six days in thermostat set at 86 °C) the dry weight were estimated d) in animals with 20 % and 30 % hyperhydration the degree of myelin deterioration was estimated e) animal locomotor activity was tested by continuous behavior tracking and analysis. Brain water content after water intoxication and following the administration of mannitol was higher than in the control group. Different degrees of hyperhydration led to different levels of brain water content and to different degrees of myelin impairment. Hyperhydration corresponding to 20 % of the body weight brought about lower locomotor activity. Increased water content in the brain after the BBB osmotic disruption is surprising because this method is frequently used in the clinical practice.
- MeSH
- hematoencefalická bariéra patofyziologie MeSH
- intoxikace vodou patofyziologie MeSH
- krysa rodu rattus MeSH
- mozek metabolismus patologie MeSH
- myelinová pochva metabolismus patologie MeSH
- osmotický tlak MeSH
- potkani Wistar MeSH
- tělesná voda metabolismus MeSH
- velikost orgánu MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Our previous experiments proved that methylprednisolone (MP) can significantly reduce axonal impairment accompanying extracellular oedema induced by the osmotic challenge (load) on the blood-brain barrier (BBB). The aim of the present work was to identify whether MP can affect myelin impairment accompanying intracellular oedema induced by water intoxication. METHODS: For induction of cellular brain oedema, the standard model of water intoxication was chosen. Animals received distilled water in amount corresponding to 15% of the animal's body weight. The volume was divided into three parts and administered intraperitoneally in 8 hours interval. Axonal changes were recognized as signs of myelin disintegration (oedematous distensions, axonal swelling, vesicles, varicosities) at histological sections stained with Black Gold and classified into four grades of myelin degradation. Hippocampal CA1 and CA3 areas and the dentate gyrus were selected for the study. Methylprednisolone was administered either intraperitoneally or intracarotically. Its effect was studied in two different time intervals: in the acute group (30 minutes after hyperhydration and MP application) and in chronic one (1 week after hyperhydration and MP application). Results: In both the acute and chronic groups, cellular oedema induced by water intoxication brought about apparent damage of myelin (compared to control animals p<0.0001). Intracarotic injection of MP was not able to influence myelin integrity changes either in the acute or in chronic group. However, intraperitoneal administration of MP increased the level of myelin deterioration in the acute group (p 0.05), but improved myelin changes in the chronic group (p<0.005). Conclusion: The effect of MP on axonal impairment during cellular brain oedema induced by water intoxication differs from that during the extracellular osmotic oedema. In the extracellular oedema, cellular metabolism is not significantly affected and myelin changes can be influenced by the neuroprotective effect of MP. The primary cause of cellular oedema is a disorder of cellular metabolism and myelin impairment is one of the structural consequences of such disorder. That is why the myelin changes are not affected by MP administration in a consistent and specific manner.
- MeSH
- axony účinky léků metabolismus patologie MeSH
- edém mozku farmakoterapie metabolismus patologie MeSH
- glukokortikoidy farmakologie MeSH
- hematoencefalická bariéra účinky léků metabolismus patologie MeSH
- intoxikace vodou farmakoterapie metabolismus patologie MeSH
- krysa rodu rattus MeSH
- methylprednisolon farmakologie MeSH
- myelinová pochva účinky léků metabolismus patologie MeSH
- nervová vlákna myelinizovaná účinky léků metabolismus patologie MeSH
- neuroprotektivní látky farmakologie MeSH
- osmotický tlak účinky léků MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
