György Sétáló

Inhibiting poly(ADP-ribose) polymerase: a potential therapy against oligodendrocyte death

Oligodendrocyte loss and demyelination are major pathological hallmarks of multiple sclerosis. In pattern III lesions, inflammation is minor in the early stages, and oligodendrocyte apoptosis prevails, which appears to be mediated at least in part through mitochondrial injury. Here, we demonstrate poly(ADP-ribose) polymerase activation and apoptosis inducing factor nuclear translocation within apoptotic oligodendrocytes in such multiple sclerosis lesions. The same morphological and molecular pathology was observed in an experimental model of primary demyelination, induced by the mitochondrial toxin cuprizone. Inhibition of poly(ADP-ribose) polymerase in this model attenuated oligodendrocyte depletion and decreased demyelination. Poly(ADP-ribose) polymerase inhibition suppressed c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation, increased the activation of the cytoprotective phosphatidylinositol-3 kinase-Akt pathway and prevented caspase-independent apoptosis inducing factor-mediated apoptosis. Our data indicate that poly(ADP-ribose) polymerase activation plays a crucial role in the pathogenesis of pattern III multiple sclerosis lesions. Since poly(ADP-ribose) polymerase inhibition was also effective in the inflammatory model of multiple sclerosis, it may target all subtypes of multiple sclerosis, either by preventing oligodendrocyte death or attenuating inflammation.

Improvement of the electron microscopic detection of peroxidase activity by means of the silver intensification of the diaminobenzidine reaction in the rat nervous system

For the detection of the peroxidase activity at the electron microscopic level, a recently developed post-intensification method is applied, which plates metallic gold onto the end-product of the diaminobenzidine (DAB) reaction. Ultrastructural analysis of rat hypoglossal neurons labeled with horseradish peroxidase (HRP) through axonal transport reveals that the method is highly specific and more sensitive than the classical HRP--DAB--OsO4 sequence. Gold grains of 2--15 nm in diameter are present in the HRP-containing organelles of the neuron, whereas other elements of the brain tissue do not contain metallic gold.

Ultrastructural characteristics of immunolabelled, corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

SummaryThe corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.

Immunohistochemical Study of the LHRH-Synthesizing Neuron System of Aged Female Rats

SummaryThe LHRH-synthesizing neuron system was studied in young proestrous and old female rats, and in aged ovariectomized or reserpine-treated females. The medial preoptic area and septal region of old animals contains more LHRH positive perikarya compared to that of young proestrous rats. Reserpine treatment moderately increases the number of immunostainable LHRH cells, while ovariectomy is ineffective in this respect.

Protective effects of the neuropeptide PACAP in diabetic retinopathy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neurotrophic and neuroprotective effects. PACAP and its receptors occur in the retina and PACAP has been applied in animal models of metabolic retinal disorders to reduce structural and functional damage. Furthermore, PACAP has been implicated as a potential anti-diabetic peptide. Our aim has been to investigate, by using a complex morphological, immunochemical and molecular biological approach, whether PACAP attenuates diabetic retinopathy. Diabetes was induced in rats with a single streptozotocin injection. PACAP was injected intravitreally into one eye (100 pmol) three times during the last week of a 3-week survival period. Retinas were processed for the following procedures: routine histology, immunohistochemistry (single and double labeling, whole-mount), quantitative reverse transcription with the polymerase chain reaction and Western blotting. Cone photoreceptors and dopaminergic amacrine and ganglion cells degenerated in diabetic retinas and glial fibrillary acidic protein were upregulated in Müller glial cells. The number of cones, the length of their outer segments and the cell number in the ganglion cell layer were decreased. PACAP ameliorated these structural changes. Moreover, PACAP increased the levels of PAC1-receptor and tyrosine-hydroxylase as detected by molecular biological methods. Thus, PACAP has significant protective effects in the diabetic retina. PACAP treatment attenuates neuronal cell loss in diabetic retinopathy, the protective effects of PACAP probably being mediated through the activation of PAC1-receptor. These results suggest that PACAP has a therapeutic potential in diabetic retinopathy.

PACAP promotes neuron survival in early experimental diabetic retinopathy.

Metabolic changes induced by diabetes lead to a multifactorial progressive disease of the retina with an extremely complex pathogenesis. One of the mechanisms of retinal cell death in diabetes is via apoptosis. Our previous results show that pituitary adenylate cyclase activating polypeptide (PACAP) attenuates the morphological and neurochemical changes in a rat model of diabetic retinopathy. The aim of this study was to investigate the mechanisms of this protective effect. Retinas of streptozotocin-induced diabetic rats were analyzed using apoptosis detection combined with immunolabeling. Western blot was used to measure levels of pro- and anti-apoptotic pathways. Intraocular PACAP injection markedly attenuated diabetic retinal injury: increased levels of the anti-apoptotic p-Akt, p-ERK1, p-ERK2, PKC, Bcl-2, while decreased levels of the pro-apoptotic p-p38MAPK and activated caspases (8, 3, 12) were detected. The number of apoptotic cells increased in all nuclear layers of diabetic retinas, but significantly decreased after PACAP treatment. Our results clearly demonstrate that the protective effects of PACAP are mediated, at least partly, by attenuating apoptosis, including also that of the dopaminergic amacrine cells. Inhibition of apoptosis is one of the PACAP-induced pathways with therapeutic potential in early experimental diabetic retinopathy.

Mitochondrial translocation of the glucocorticoid receptor in double-positive thymocytes correlates with their sensitivity to glucocorticoid-induced apoptosis

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.

Prohepcidin binds to the HAMP promoter and autoregulates its own expression.

Hepcidin is the major regulatory peptide hormone of iron metabolism, encoded by the HAMP (hepcidin antimicrobial peptide) gene. Hepcidin is expressed mainly in hepatocytes, but is also found in the blood in both a mature and prohormone form. Although, the function of mature hepcidin and the regulation of the HAMP gene have been extensively studied, the intracellular localization and the fate of prohepcidin remains controversial. In the present study, we propose a novel role for prohepcidin in the regulation of its own transcription. Using indirect immunofluorescence and mCherry tagging, a portion of prohepcidin was detected in the nucleus of hepatocytes. Prohepcidin was found to specifically bind to the STAT3 (signal transducer and activator of transcription 3) site in the promoter of HAMP. Overexpression of prohepcidin in WRL68 cells decreased HAMP promoter activity, whereas decreasing the amount of prohepcidin caused increased promoter activity measured by a luciferase reporter-gene assay. Moreover, overexpression of the known prohepcidin-binding partner, α-1 antitrypsin caused increased HAMP promoter activity, suggesting that only the non-α-1 antitrypsin-bound prohepcidin affects the expression of its own gene. The results of the present study indicate that prohepcidin can bind to and transcriptionally regulate the expression of HAMP, suggesting a novel autoregulatory pathway of hepcidin gene expression in hepatocytes.

Frontal deafferentation of the mediobasal hypothalamus in the neonatal rat and its effects on the preoptico-infundibular LHRH-tract

SummaryBy use of the peroxidase-antiperoxidase-complex (PAP) immunohistological method, the preoptico-infundibular LHRH-tract was studied in adult female rats in which frontal hypothalamic deafferentation was performed at the third or tenth postnatal day. In the former group, this LHRH-tract appeared to be similar to that of the intact controls; the animals showed regular vaginal cycles and ova were present in their oviducts. In the latter group, however, marked reduction in the number of the LHRH-nerve fibers was observed behind the sites of the deafferentation in the mediobasal hypothalamus (MBH), whereas LHRH-immunoreactive perikarya and nerve fibers containing the immunoreactive material were seen rostral to the plane of severance. In these animals reduction of LHRH-fibers in the MBH was accompanied by an anovulatory syndrome characterized by constant vaginal cornification and polyfollicular ovaries. Comparing the glial scar formation induced by the cut, significant differences were detected between the two experimental groups. In the animals deafferented on the 3rd day of life, reduction of nerve cells was seen along the cut, but LHRH-fibers crossing the thin glial scar were detectable in large numbers. On the other hand, in the animals deafferented on the 10th postnatal day, extensive glial scar tissue appeared to interrupt the LHRH-fibers rostral to the cut.

TRPA1 deficiency is protective in cuprizone-induced demyelination—A new target against oligodendrocyte apoptosis

Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long‐term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone‐induced demyelination. This toxin‐induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone‐induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen‐activated protein kinase pathways, as well as transcription factor c‐Jun and a proapoptotic Bcl‐2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca2+ concentration modulate astrocyte functions, and influence the pro or anti‐apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166–2180