Targeting myelin lipid metabolism as a potential therapeutic strategy in a model of CMT1A neuropathy.

In patients with Charcot-Marie-Tooth disease 1A (CMT1A), peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. Here, we show that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo. This treatment rescues the number of myelinated axons in the peripheral nerves of the CMT rats and leads to a marked amelioration of neuropathic symptoms. We propose that lipid supplementation is an easily translatable potential therapeutic approach in CMT1A and possibly other dysmyelinating neuropathies.

Decreased microglial numbers in Vav1-Cre+:dicer knock-out mice suggest a second source of microglia beyond yolk sac macrophages.

Microglia represent the resident macrophages of the central nervous system (CNS). While it is clear that microglia recruitment is established by differentiation of primitive yolk sac (YS) macrophages and consecutive invasion of the brain, starting around E8 in rodents (Ginhoux et al., 2010), more recent studies suggest that a non-YS contribution to the microglia population should not entirely be dismissed (Swinnen et al., 2013; Xu et al., 2015). Therefore, we used Vav1-Cre+:dicer knock-out mice in order to study the effect of the post-YS hematopoiesis on the definitive microglial population in late prenatal (E16.5, E18.5) and early postnatal brains (P0, P1). Since Vav1 is thereby exclusively expressed in hematopoietic cells starting at E11, the depletion of the micro RNA processing enzyme dicer in Vav1-positive cells allows interfering with post-YS microglia recruitment. Using this approach, analysis of the number of Iba-1 positive microglia revealed a reduction of microglial numbers by 40% in knock-out mice at P1 compared to their individual control littermates. Noteworthy, immunolabeling for Ki-67 and active caspase 3 confirmed that the differences in the microglial numbers are not related to differential rates of proliferation or apoptosis. Therefore, our data demonstrates that interfering with the definitive hematopoiesis highly impacts on the microglial population, implicating an important role of post-YS hematopoiesis on microglial development and recruitment.

Autophagy in adipose tissue of patients with obesity and type 2 diabetes.

BACKGROUND: Pathophysiology of obesity is closely associated with enhanced autophagy in adipose tissue (AT). Autophagic process can promote survival or activate cell death. Therefore, we examine the occurrence of autophagy in AT of type 2 diabetes (T2D) patients in comparison to obese and lean individuals without diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Numerous autophagosomes accumulated within adipocytes were visualized by electron transmission microscopy and by immunofluorescence staining for autophagy marker LC3 in obese and T2D patients. Increased autophagy was demonstrated by higher LC3-II/LC3-I ratio, up-regulated expression of LC3 and Atg5 mRNA, along with decreased p62 and mTOR protein levels. Increased autophagy occurred together with AT inflammation. CONCLUSIONS: Our data suggest fat depot-related differences in autophagy regulation. In subcutaneous AT, increased autophagy is accompanied by increased markers of apoptosis in patients with obesity independently of T2D. In contrast, in visceral AT only in T2D patients increased autophagy was related to higher markers of apoptosis.

Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.

BACKGROUND: Human head and neck squamous cell carcinoma (HNSCC) fundamentally vary in their susceptibility to different cytotoxic drugs and treatment modalities. There is at present no clinically accepted test system to predict the most effective therapy for an individual patient. METHODS: Therefore, we established tumour-derived slice cultures which can be kept in vitro for at least 6 days. Upon treatment with cisplatin, docetaxel and cetuximab, slices were fixed and paraffin sections were cut for histopathological analysis. RESULTS: Apoptotic fragmentation, activation of caspase 3, and cell loss were observed in treated tumour slices. Counts of nuclei per field in untreated compared with treated slices deriving from the same tumour allowed estimation of the anti-neoplastic activity of individual drugs on an individual tumour. CONCLUSION: HNSCC-derived slice cultures survive well in vitro and may serve not only to improve personalised therapies but also to detect mechanisms of tumour resistance by harvesting surviving tumour cells after treatment.

Resveratrol and desferoxamine protect human OxLDL-treated granulosa cell subtypes from degeneration.

CONTEXT: Obese women suffer from anovulation and infertility, which are driven by oxidative stress caused by increased levels of lipid peroxides and circulating oxidized low-density lipoprotein (oxLDL). OxLDL binds to lectin-like oxLDL receptor 1 (LOX-1), cluster of differentiation 36 (CD36), and toll-like receptor 4 (TLR4) and causes cell death in human granulosa cells (GCs). OBJECTIVE: Our objective was to reveal whether treatment with antioxidants resveratrol (RES) and/or desferoxamine (DFO) protect GCs from oxLDL-induced damage. DESIGN AND SETTING: This basic research study was performed at the Institute of Anatomy and the Clinic of Reproductive Medicine. PATIENTS: Patients were women undergoing in vitro fertilization therapy. MAIN OUTCOME MEASURES: GC cultures were treated with oxLDL alone or with RES or DFO under serum-free conditions for up to 36 hours. Dead cells were determined by propidium iodide uptake, cleaved caspase-3 expression, and electron microscopy. Mitosis was detected by Ki-67 immunostaining. LOX-1, TLR4, CD36, and heat-shock protein 60 were examined by Western blot. Measurement of oxidative stress markers (8-iso-prostaglandin F2α, advanced glycation end products, and protein carbonyl content) was conducted with ELISA kits. RESULTS: Different subtypes of human GCs exposed to RES or DFO were protected as evidenced by the lack of cell death, enhanced mitosis, induction of protective autophagy, reduction of oxidative stress markers, and reduced expression of LOX-1, TLR4, CD36, and heat-shock protein 60. Importantly, RES could restore steroid biosynthesis in cytokeratin-positive GCs, which exhibited significant induction of steroidogenic acute regulatory protein. CONCLUSIONS: RES and DFO exert a protective effect on human GCs. Thus, RES and DFO may help improve the treatment of obese women or polycystic ovarian syndrome patients undergoing in vitro fertilization therapy.

Increased autophagy in peripheral nerves may protect Wistar Ottawa Karlsburg W rats against neuropathy.

OBJECTIVE: Wistar Ottawa Karlsburg W (RT1(u)) rats (WOKW) develop obesity, dyslipidemia, moderate hypertension, hyperinsulinemia and impaired glucose tolerance prone to induce peripheral neuropathy (PN). Autophagy has been shown to prevent neurodegeneration in the central and peripheral nervous system. We analyzed the potential protective role of autophagy in an established rat model in preventing PN. METHODS: We examined electrophysiology (motor-and sensory/mixed afferent conduction velocities and the minimal F-wave latency) and morphology, including ultrathin sections, myelin sheath thickness (g-ratio) and immunohistochemical markers of autophagy and inflammation in the sciatic nerve of five-month-old, male WOKW as compared to Wistar derived, congenic LEW.1W control rats, characterized by the same major histocompatibility complex as WOKW rats (RT1(u)). Moreover, the expression of axonal and synaptic proteins (NF68, GAP43, MP0), autophagy- (Atg5, Atg7, LC3), and apoptosis (cleaved caspase-3)-related markers was measured using Western blot. RESULTS: No abnormalities in nerve electrophysiology and morphology were found in WOKW compared to LEW.1W rats. However, autophagosomes were more frequently apparent in sciatic nerves of WOKW rats. In Western blot analyses no significant differences in expression of neuronal structural proteins were found, but autophagy markers were up-regulated in WOKW compared to LEW.1W sciatic nerves. Immunostaining revealed a greater infiltration of Iba1/ED-1-positive macrophages, CD-3-positive T-cells and LC3-expression in sciatic nerves of WOKW rats. CONCLUSIONS: Our results indicate that WOKW rats show an up-regulated autophagy and a mild inflammatory response but do not develop overt neuropathy. We suggest that autophagy and inflammatory cells may exert a protective role in preventing neuropathy in this rat model of the metabolic syndrome but the mechanism of action is still unclear.

Bone marrow transplantation improves the outcome of Atm-deficient mice through the migration of ATM-competent cells.

Ataxia telangiectasia (A-T) is a highly pleiotropic disorder. Patients suffer from progressive neurodegeneration, severe bronchial complications, immunodeficiency, hypersensitivity to radiotherapy and elevated risk of malignancies. Leukemia and lymphoma, along with lung failure, are the main causes of morbidity and mortality in A-T patients. At present, no effective therapy for A-T exists. One promising therapeutic approach is bone marrow transplantation (BMT) that is already used as a curative therapy for other genomic instability syndromes. We used an established clinically relevant non-myeloablative host-conditioning regimen and transplanted green fluorescent protein (GFP)-expressing ataxia telangiectasia mutated (ATM)-competent bone marrow-derived cells (BMDCs) into Atm-deficient mice. GFP expression allowed tracking of the potential migration of the cells into the tissues of recipient animals. Donor BMDCs migrated into the bone marrow, blood, thymus, spleen and lung tissue of Atm-deficient mice showing an ATM-competent phenotype. BMT inhibited thymic lymphomas, normalized T-lymphocyte populations, improved weight gain and rearing activity of Atm-deficient mice. In contrast, no GFP(+) cells were found in the cerebellum or cerebrum, and we detected decreased size index in MRI imaging of the cerebellum in 8-month-old transplanted Atm-deficient mice in comparison to wild-type mice. The repopulation with ATM-competent BMDCs is associated with a prolonged lifespan and significantly improved the phenotype of Atm-deficient mice.

Involvement of the auditory brainstem system in spinocerebellar ataxia type 2 (SCA2), type 3 (SCA3) and type 7 (SCA7).

AIMS: The spinocerebellar ataxia type 2 (SCA2), type 3 (SCA3) and type 7 (SCA7) are clinically characterized by progressive and severe ataxic symptoms, dysarthria, dysphagia, oculomotor impairments, pyramidal and extrapyramidal manifestations and sensory deficits. Although recent clinical studies reported additional disease signs suggesting involvement of the brainstem auditory system, this has never been studied in detail in SCA2, SCA3 or SCA7. METHODS: We performed a detailed pathoanatomical investigation of unconventionally thick tissue sections through the auditory brainstem nuclei (that is, nucleus of the inferior colliculus, nuclei of the lateral lemniscus, superior olive, cochlear nuclei) and auditory brainstem fibre tracts (that is, lateral lemniscus, trapezoid body, dorsal acoustic stria, cochlear portion of the vestibulocochlear nerve) of clinically diagnosed and genetically confirmed SCA2, SCA3 and SCA7 patients. RESULTS: Examination of unconventionally thick serial brainstem sections stained for lipofuscin pigment and Nissl material revealed a consistent and widespread involvement of the auditory brainstem nuclei in the SCA2, SCA3 and SCA7 patients studied. Serial brainstem tissue sections stained for myelin showed loss of myelinated fibres in two of the auditory brainstem fibre tracts (that is, lateral lemniscus, trapezoid body) in a subset of patients. CONCLUSIONS: The involvement of the auditory brainstem system offers plausible explanations for the auditory impairments detected in some of our and other SCA2, SCA3 and SCA7 patients upon bedside examination or neurophysiological investigation. However, further clinical studies are required to resolve the striking discrepancy between the consistent involvement of the brainstem auditory system observed in this study and the comparatively low frequency of reported auditory impairments in SCA2, SCA3 and SCA7 patients.

Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments.

Spinocerebellar ataxia type 7 (SCA7) represents a rare and severe autosomal dominantly inherited ataxic disorder and is among the known CAG-repeat, or polyglutamine, diseases. In contrast to other currently known autosomal dominantly inherited ataxic disorders, SCA7 may manifest itself with different clinical courses. Because the degenerative changes evolving during these different clinical courses are not well known, many neurological disease symptoms still are unexplained. We performed an initial pathoanatomical study on unconventional thick tissue sections of the brain of a clinically diagnosed and genetically confirmed adult-onset SCA7 patient with progressive visual impairments. In this patient we observed loss of myelinated fibres in distinct central nervous fibre tracts, and widespread degeneration of the cerebellum, telencephalon, diencephalon and lower brainstem. These degenerative changes offer appropriate explanations for a variety of less-understood neurological symptoms in adult-onset SCA7 patients with visual impairments: gait, stance and limb ataxia, falls, dysarthria, dysphagia, pyramidal signs, Parkinsonian features, writing problems, impairments of saccades and smooth pursuits, altered pupillary functions, somatosensory deficits, auditory deficits and mental impairments.

Self-tolerance in the immune privileged CNS: lessons from the entorhinal cortex lesion model.

Upon peripheral immunization with myelin epitopes, susceptible rats and mice develop T cell-mediated demyelination similar to that observed in the human autoimmune disease multiple sclerosis (MS). In the same animals, brain injury does not induce autoimmune encephalomyelitis despite massive release of myelin antigens and early expansion of myelin specific T cells in local lymph nodes, indicating that the self-specific T cell clones are kept under control. Using entorhinal cortex lesion (ECL) to induce axonal degeneration in the hippocampus, we identified possible mechanisms of immune tolerance after brain trauma. Following ECL, astrocytes upregulate the death ligand CD95L, allowing apoptotic elimination of infiltrating activated T cells. Myelin-phagocytosing microglia express MHC-II and the costimulatory molecule CD86, but lack CD80, which is found only on activated antigen presenting cells (APCs). Restimulation of invading T cells by such immature APCs (e.g. CD80 negative microglia) may lead to T cell anergy and/or differentiation of regulatory/Th3-like cells due to insufficient costimulation and presence of high levels of TGF-beta and IL-10 in the CNS. Thus, T cell -apoptosis, -anergy, and -suppression apparently maintain immune tolerance after initial expansion of myelin-specific T lymphocytes following brain injury. This view is supported by a previous metastatistical analysis which rejected the hypothesis that brain trauma is causative of MS (Goddin et al., 1999). However, concomitant trauma-independent proinflammatory signals, e.g., those evoked by clinically quiescent infections, may trigger maturation of APCs, thus shifting a delicate balance from immune tolerance and protective immune responses to destructive autoimmunity.

Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

Differential expression of costimulatory molecules B7-1 and B7-2 on microglial cells induced by Th1 and Th2 cells in organotypic brain tissue.

Autoreactive T-cells are involved in demyelination, neurodegeneration, and the recruitment of peripheral macrophages and nonspecific activated T-cells in autoimmune diseases such as multiple sclerosis (MS). The ligation of costimulatory B7 molecules on microglia with CD28/CTLA-4 on T-cells is thought to be crucial to the onset and course of MS and its rodent model experimental autoimmune encephalomyelitis (EAE). It is currently unclear as to how far the nature of infiltrating T-cells has an impact on the expression of the B7 molecules on microglia, the resident antigen-presenting cells (APCs) of the brain. We studied the expression of B7-1 and B7-2 on microglia after encounter with preactivated Th1 and Th2 cells from transgenic mice whose T-cells express a receptor (TCR) either specific to myelin basic protein (MBP) or ovalbumin (OVA) using murine organotypic entorhinal-hippocampal slice cultures (OEHSC). Our main finding was that Th1 cells downregulate the constitutive expression of B7-2 and induce B7-1 expression while Th2 cells do not induce this B7-1 upregulation. The main difference between MBP- and OVA-specific cells was seen in experiments were Th1 cells had direct contact to APCs but not to brain tissue. In contrast to MBP-specific Th1 cells, OVA-specific Th1 cells required the addition of antigen to upregulate B7-1 and downregulate B7-2. When the cells were allowed to have contact to brain tissue, no difference was seen in the pattern of B7 regulation between OVA- and MBP-specific T-cells. Our data suggest that T-cells are able to modulate B7 expression on microglial cells in the brain independent of antigen presentation through TCR/MHC-II ligation but presumably by soluble mediators.

Comparison of major and trace element concentrations in Danish greenhouse tomatoes (Lycopersicon esculentum Cv. Aromata F1) cultivated in different substrates.

The concentration of major and trace elements was determined for tomato (Lycopersicon esculentumcv. Aromata F1) fruits grown in three different substrate systems. The systems were soil and rockwool irrigated with a normal nutrient solution and rockwool irrigated with a nutrient solution with elevated electrical conductivity (EC). At three harvest times, tomato fruits were analyzed for Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Sr, and Zn by ICP-AES and for Cd, Cr, Mo, Ni, Pb, Sn, and V by HR-ICPMS. The concentrations of Ca, Cd, Fe, Mn, Mo, Na, Ni, Sr, and Zn were significantly different (p < 0.05) for tomato fruits grown on the different substrates. Between the harvest times different levels (p < 0.05) were shown for Ca, Cd, Fe, Mn Na, Ni, Sr, Zn Cu, K, Mg, P, Sn, and V. The concentration of Cd was >15 times higher and the concentration of Ca was 50-115% higher in soil-grown fruits than in rockwool-grown fruits. Principal component analysis applied on each harvest split the data into two groups. One group includes soil-grown fruits, and the other group includes rockwool-grown fruits with the two different nutrient solutions.

Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat.

Traumatic brain injury is followed by increased extracellular glutamate concentration. Uptake of glutamate is mainly mediated by the glial glutamate transporters GLAST and GLT-1. Extent and distribution of GLAST and GLT-1 were studied in a rat model of controlled cortical impact injury (CCII). Western Blot analysis revealed lowest levels of GLAST and GLT-1 with a decrease by 40%-54% and 42%-49% between 24 and 72 h posttrauma. By 8 h after CCII, CSF glutamate levels were increased (10.5 microM vs. 2.56 microM in controls; P < 0.001), reaching maximum values by 48 h. A significant increase in de novo GLAST and GLT-1 expressing ramified microglia was observed within 4 h, reached a stable level by 48 h, and remained high up to 72 h after CCII. Furthermore, ramified microglia de novo expressed the neuronal glutamate transporter EAAC1 after CCII. Following CCII, GLAST/GLT-1 and GFAP coexpressing astrocytes were immediately reduced, reaching minimum levels within 8 h. This reduction of expression could be either due to protein downregulation or loss of astrocytes. At 72 h, a marked population of GLAST- and GLT-1-positive reactive astrocytes appeared. These results support the hypothesis that reduced astrocytic GLAST and GLT-1 protein levels following CCII contribute to evolving secondary injury. Microglia are capable of de novo expressing glutamate transporter proteins, indicating that the expression of glial and neuronal glutamate transporters is not restricted to a specific glial or neuronal lineage. Ramified microglia may play an important compensatory role in the early regulation of extracellular glutamate after CCII.

Th2 cells support intrinsic anti-inflammatory properties of the brain.

In experimental autoimmune encephalomyelitis (EAE), Th1 cells are responsible for disease induction while Th2 cells can be protective. To address the mechanisms of this differential behavior, we utilized organotypic murine entorhinal-hippocampal slice cultures to analyze interactions between myelin basic protein-specific Th1 and Th2 cells with microglial cells. While both Th1 and Th2 cells induced CD40 expression, only Th1 cells induced intercellular adhesion molecule-1 (ICAM-1) expression on microglia. Moreover, Th2 cells prevented or even reversed Th1-induced ICAM-1 upregulation. Evidently, Th2 cells could diminish Th1-induced inflammatory reactions and actively support the resting state of microglia, which could be one mechanism of Th2-mediated remission of neuroinflammation during EAE.

Turnover of rat brain perivascular cells.

Brain perivascular spaces harbor a population of cells which exhibit high phagocytic capacity. Therefore, these cells can be labeled by intraventricular injection of tracers. Such perivascular cells at the interface between blood and brain are believed to belong to the monocyte/macrophage lineage and to be involved in antigen presentation. Currently, it is unclear whether these cells undergo a continuous turnover by entering and leaving the bloodstream. Using bone-marrow-chimeric animals, migration of donor macrophages into brain perivascular spaces has been reported. On the other hand, following intracerebral injection of india ink into nontransplanted animals, ink-labeled perivascular cells were still found 2 years after injection, suggesting a high stability of this cell pool. Thus, the turnover of perivascular cells observed in chimeras might be a result of bone marrow transplantation rather than a physiological occurrence. To address this issue, we monitored de novo invasion of macrophages into perivascular spaces of apparently healthy adult rats by applying techniques other than bone marrow transplantation, (i) consecutive injections of different tracers and (ii) ex vivo isolation of macrophages from the blood, cell labeling, and reinjection into the same animal to avoid MHC mismatch. Both approaches revealed vivid de novo invasion of macrophages into perivascular spaces, but not into brain parenchyma, rendering untenable the concept of perivascular cells forming a stable population of macrophages in the brain. Thus, brain perivascular spaces are under permanent immune surveillance of blood borne macrophages in normal adult rats.

Plasticity following lesion: help and harm from the immune system.

In contrast to other organs where the tissue is capable of replacing lost cells and thus regaining tissue function, immune cell recruitment and activation is suppressed in the CNS in order to minimize secondary damage after lesion. This state of immune privilege has its cost because the injured tissue cannot fully benefit from growth-promoting effects accompanying inflammatory responses. These responses include phagocytosis of growth-inhibiting myelin debris by cells of the innate immune system and the recently described protection of surviving fibers by myelin-specifie T cells of the adaptive immune system. While the signals suppressing macrophage functions in the CNS are yet to be defined, it seems that help from T cells is diminished by apoptosis-induction via death-inducing ligands. Indeed, the death ligand CD95L (FasL, APO 1 L) is constitutively found on neurons, microglia and astrocytes. Its upregulation on astrocytes during axonal degeneration in the hippocampus after entorhinal lesion is accompanied by the appearance of apoptotic leukocytes. T cells also express CD95L and TNF-related apoptosis- inducing ligand (TRAIL), and the presence of CD95 (Fas, APOI) and TRAIL-receptors renders brain cells putative targets of T cell-induced apoptosis. Thus, blockade of death ligands could be helpful by simultaneously enhancing T cell survival and blocking T cell-mediated brain cell death. This is only one example of how boosting helpful immune cell functions and abrogating their destructive effects might help to overcome the brain's failure to regenerate after axonal lesions.