Redox modulation of stress resilience by Crocus sativus L. for potential neuroprotective and anti-neuroinflammatory applications in brain disorders: From molecular basis to therapy.

Recent evidence demonstrates that Crocus sativus L. (saffron) counteracts oxidative stress, mitochondrial dysfunction and neuroinflammation, closely linked to initiation and progression of major brain pathologies. Interestingly, saffron constituents such as crocin, crocetin and safranal can exert antioxidant or toxic effects depending on their endogenous concentration. According to the hormesis principles, at low dose they act as antioxidants in a wide range of brain diseases by upregulating Nrf2 signaling pathway and the expression of vitagenes, such as NAD(P)H-quinone oxidoreductase (NQO1), glutathione transferase (GT), heme oxygenase-1 (HO-1), sirtuin-1 (Sirt1) and thioredoxin (Trx) system. Importantly, neuronal dysregulation of Nrf2 pathway can be a prominent cause of selective susceptibility, under neuroinflammatory conditions, due to the high vulnerability of brain cells to oxidative stress. Here we discuss natural inducers from saffron targeting Nrf2/vitagene pathway for development of new therapeutical strategies to suppress oxidative stress and neuroinflammation and consequently cognitive dysfunction. In this review we also focus on the hormetic effect of saffron active constituents, summarizing their neuroprotective and anti-neuroinflammatory properties, as well as pharmacological perspectives in brain disorders.

Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes.

Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status a cellular adaptive response occurs requiring functional chaperones, antioxidant production and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring plasma reduced and oxidized glutathione, as well as pentosidine, protein carbonyls lipid oxidation products 4-hydroxy-2-nonenal and F2-isoprostanes in plasma, and lymphocytes, whereas the lymphocyte levels of the heat shock proteins (HSP) HO-1, Hsp72, Sirtuin-1, Sirtuin-2 and thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. Plasma GSH/GSSG showed a significant decrease in type 2 diabetes as compared to control group, associated with increased pentosidine, F2-isoprostanes, carbonyls and HNE levels. In addition, lymphocyte levels of HO-1, Hsp70, Trx and TrxR-1 (P<0.05 and P<0.01) in diabetic patients were higher than in normal subjects, while sirtuin-1 and sirtuin-2 protein was significantly decreased (p<0.05). In conclusion, patients affected by type 2 diabetes are under condition of systemic oxidative stress and, although the relevance of downregulation in sirtuin signal has to be fully understood, however induction of HSPs and thioredoxin protein system represent a maintained response in counteracting systemic pro-oxidant status. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex.

BACKGROUND: Gap junctions are specialized channels composed of several connexins, membrane proteins that mediate electrical and metabolic coupling between cells. Previous data have suggested that changes in the expression of Cx43, the main astrocytic Cx isoform, may be involved in seizure activity in human epileptic tissue. However, Cx43 has never been examined in focal cortical dysplasia (FCD) and in other human refractory epilepsies. METHODS: We analyzed Cx43 protein localization and Cx43 mRNA levels in surgical specimens of cortex from a cohort of patients with intractable epilepsy vs control nonepileptic tissue. Samples had neuropathologically defined diagnosis of cryptogenic epilepsy or epilepsy secondary to FCD. RESULTS: Cx43 immunoreactivity, which labeled punctate elements, did not reveal distinctive features in cryptogenic epilepsy and FCD type IA and IIA. A peculiar pattern of immunolabeling was instead observed in FCD type IIB, in which large aggregates of Cx43-immunopositive puncta were clustered around subsets of balloon cells and astrocytes. Further characterization revealed that these balloon cells do not express markers of precursor cells, such as CD34. Quantitative real-time reverse transcriptase PCR showed elevated levels of Cx43 transcript in a subgroup (25%) of cryptogenic epilepsy specimens compared to control and FCD ones. CONCLUSIONS: Our study points out that a rearrangement of Cx43-positive elements is part of abnormal tissue organization in FCD type IIB, and that cryptogenic epilepsies include forms with increased Cx43 mRNA expression. The data implicate functional consequences of altered Cx43 expression, and therefore of altered gap junctional coupling, in abnormal network properties of subtypes of human refractory epilepsies.

Regulation of connexin gene expression during skeletal muscle regeneration in the adult rat.

In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24 h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesion-forming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24 h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers.

Expression of the rat connexin 39 (rCx39) gene in myoblasts and myotubes in developing and regenerating skeletal muscles: an in situ hybridization study.

We report a detailed analysis of the expression pattern of the recently identified rat connexin gene, named rat connexin 39 (rCx39), both during embryonic development and in adult life. Qualitative and quantitative reverse transcription/polymerase chain reaction analysis showed intense expression of rCx39 restricted to differentiating skeletal muscles, with a peak of expression detected at 18 days of embryonic life, followed by a rapid decline to undetectable levels within the first week of postnatal life. A combination of the in situ hybridization technique for the detection of rCx39 mRNA and immunohistochemistry for myogenin, a myoblast-specific marker, allowed us to establish that the mRNA for this connexin was expressed in myogenin-positive myoblasts and early myotubes but disappeared in mature myotubes. Moreover, in adult animals, rCx39 mRNA was expressed in myogenic cells involved in skeletal myofiber regeneration following a crush injury. This is the first case of a connexin being mainly expressed in the myogenic cell lineage. The information presented should pave the way to novel molecular approaches in studies on the role of connexin-based gap-junctional communication in skeletal muscle differentiation and regeneration.

Connexin-30 mRNA is up-regulated in astrocytes and expressed in apoptotic neuronal cells of rat brain following kainate-induced seizures.

Glial connexins (Cxs) make an extensively interconnected functional syncytium created by a network of gap junctions between astrocytes and oligodendrocytes. Among Cxs expressed in the brain, Cx30 is expressed in grey matter astrocytes, as shown at the protein level by immunoistochemistry. In the present study we aimed to perform a detailed study of the regional distribution of Cx30 mRNA in the adult and postnatal developing rat brain, analyzing its expression by in situ hybridization, and determining its cell type localization by double labeling. Recently, it has been suggested that neuronal activity may control the level of intercellular communication between astrocytes through gap junctions channels. Thus, a second aim of the present study was to investigate the short-term effects of kainate-induced seizures on Cx30 expression. The results showed that, in basal condition, Cx30 was expressed only in grey matter astrocytes with distinct regional patterns in developing and adult brain. Kainate treatment induced strong and region-specific changes of astroglial Cx30 mRNA levels and expression of Cx30 mRNA in neuronal cells undergoing cell death, suggesting a direct or indirect involvement of this connexin in the neuronal apoptotic process.

Identification and functional expression of HCx31.9, a novel gap junction gene.

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.

Cx36 and the function of endocrine pancreas.

The secretory, duct, connective and vascular cells of pancreas are connected by gap junctions, made of different connexins. The insulin-producing beta-cells, which form the bulk of endocrine pancreatic islets, express predominantly Cx36. To assess the function of this connexin, we have first studied its expression in rats, during sequential changes of pancreatic function which were induced by the implantation of a secreting insulinoma. We observed that changes in beta-cell function were paralleled by changes in Cx36 expression. We have also begun to investigate mutant mice lacking Cx36. The absence of this protein did not affect the development and differentiation of beta-cells but appeared to alter their secretion. We have studied this effect in MIN6 cells which spontaneously express Cx36. After stable transfection of a construct that markedly reduced the expression of this connexin, we observed that MIN6 cells were no more able to secrete insulin, in contrast to wild type controls, and differentially displayed a series of still unknown genes. The data provide evidence that Cx36-dependent signaling contributes to regulate the function of native and tumoral insulin-producing cells.

Cx36 preferentially connects beta-cells within pancreatic islets.

Previous studies have provided evidence for the transcripts of Cx43 and Cx45 within pancreatic islets. As of yet, however, it has proven difficult to unambiguously demonstrate the expression of these proteins by islet cells. We have investigated whether Cx36, a new connexin species recently identified in mammalian brain and retina, may also be expressed in pancreatic islets. Using probes that permitted the original identification of Cx36 in the central nervous system, we show that a transcript for Cx36 is clearly detectable in rat pancreatic islets. Using novel and affinity-purified polyclonal antibodies, we have found that Cx36 is actually expressed in pancreatic islets. Both in situ hybridization and immunolabeling indicated that this connexin is abundant in the centrally located insulin-producing beta-cells and is expressed much less, if at all, by the other endocrine cell types. This differential expression was further confirmed on fluorescence-activated cell sorter-purified preparations enriched in either beta- or non-beta-cells. The finding of a differential distribution of Cx36 within distinct regions of pancreatic islets creates the possibility that this connexin may provide the establishment of selective pathways of communication between the different types of endocrine cells comprising the pancreatic islet.

Expression of connexin36 in the adult and developing rat brain.

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.

Expression of Cx36 in mammalian neurons.

Cx36 is the first mammalian member of a novel subgroup of the connexin family, characterized by a long cytoplasmic loop, a peculiar gene structure and a preferential expression in cell types of neural origin. In the present review we summarize the evidence in favour of its predominant expression in neuronal cells in the mammalian central nervous system, such as results from experiments with specific neurotoxins and co-localization of Cx36 mRNA and a neuronal marker. We also report a detailed description of Cx36 mRNA distribution in the rat and human central nervous system by in situ hybridization and, for each brain region, we correlate the novel findings with previous morphological or functional demonstrations of neuronal gap junctions in the same area.

Central motor conduction to lower limb after transcranial magnetic stimulation in spinocerebellar ataxia type 2 (SCA2).

OBJECTIVES: To evaluate central motor conduction to lower limbs in spinocerebellar ataxia type 2 (SCA2). METHODS: Transcranial magnetic stimulation was performed to study the corticospinal tracts of 18 patients with SCA2. RESULTS: Central motor conduction time (CMCT) to lower limbs and thresholds were abnormal in 8 patients (44%); CMCT and thresholds were significantly correlated with disease duration and disability. CONCLUSIONS: Corticospinal tract involvement is more frequent than previously reported in SCA2. Prolonged CMCT and increased threshold should not be used to differentiate between various type of autosomal dominant cerebellar ataxia. Similar to that reported in Friedreich's ataxia, we suggest that examining central motor conduction to the lower limbs may assist in evaluating the progressive steps of neurodegeneration in SCA2.

Identification of SCA2 mutation in cases of spinocerebellar ataxia with no family history in mid-eastern Sicily.

Differential diagnosis between autosomal dominant cerebellar ataxia type I (ADCA I) and idiopathic cerebellar ataxia type P (IDCA-P) is very difficult given only clinical and neuroradiological data. The only certain distinctive characteristic is the presence or absence of family history. We observed 7 patients with late-onset cerebellar ataxia associated with other non-cerebellar signs and without a family history of the disease in which clinical signs were comparable to symptoms found in SCA2. The neuroradiological study showed olivopontocerebellar atrophy in all patients and the presence of hyperintensity of the transverse pontine fibers in 6 patients (85. 6%); molecular analysis showed SCA2 mutations in 2 patients. We also report the case of a patient who was initially considered as IDCA-P but who was later correctly identified as SCA2 with an atypical family history (false IDCA-P), after a genetic mutation was found and following an interview with the mother. Our data suggest that spinocerebellar ataxia syndrome should be defined as idiopathic not only after having excluded the possible symptomatic causes but also in the absence of family history, after having excluded the presence of genetic mutation. We believe that family history, in late-onset spinocerebellar ataxia, cannot be considered as the differential criterion among hereditary (ADCA-I) and non-hereditary (IDCA-P) forms; molecular analysis is required for a correct diagnosis.

Structure, chromosomal localization, and brain expression of human Cx36 gene.

Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the gene structure is that typical of the Cx35/36 subgroup observed in the other species (presence of a single intron located within the coding region, 71 bp after the translation initiation site). The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal among the cerebral areas examined by in situ hybridization was observed in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling was also observed in several myelencephalic nuclei, in specific cells of the the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus, and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells were revealed in all the lamina of the spinal cord gray matter. The chromosomal localization of the human Cx36 gene was determined by fluorescence in situ hybridization. The results allowed assignment of the gene to band 15q14, thus making it a possible candidate gene for a form of familial epilepsy previously linked to the same chromosomal band. The knowledge of the human Cx36 gene sequence, of its chromosomal localization, and of its pattern of expression opens new avenues for the analysis of its possible involvement in human genetic and acquired neuropathology.

Supratentorial atrophy in spinocerebellar ataxia type 2: MRI study of 20 patients.

There have been only few studies of brain magnetic resonance imaging (MRI) in spinocerebellar ataxia (SCA) type 2. We investigated 20 SCA2 patients, from 11 Sicilian families, and 20 age-matched control subjects using MRI. Our data confirm that olivopontocerebellar atrophy (OPCA) is the typical pattern in SCA2. We found no significant correlation between infratentorial atrophy, disease duration, or the number of CAG repeats in our SCA2 patients, but there was supratentorial atrophy in 12 patients, with a significant correlation between supratentorial atrophy and disease duration. OPCA appears to represent the "core" of the SCA2: however, central nervous system involvement is not limited to pontocerebellar structures. We therefore consider central nervous system degeneration in SCA2 as a widespread atrophy. MRI is helpful in diagnosing SCA, but it is not diagnostic in the absence of clinical and molecular studies. We suggest that serial MRI may play a role in evaluating "in vivo" the progressive steps of neurodegeneration in SCA2, for a better comprehension of the pathophysiology of this disorder.

Clinical and molecular analysis of 11 Sicilian SCA2 families: influence of gender on age at onset.

Autosomal dominant cerebellar ataxias (ADCAs) are a complex group of slowly progressive neurodegenerative disorders characterized by gait and stance ataxia, dysarthria and other symptoms of nervous system involvement. ADCA type I is the commonest form and is genetically heterogeneous; several loci have been identified. Spinocerebellar ataxia type 2 (SCA2) has been mapped to chromosome 12, with expanded cytosine-adenine-guanine (CAG) repeats being identified as the mutational cause of the disease. We investigated 15 families, all originating from mid-eastern Sicily, with ADCA type I; molecular studies performed in 12 families showed the SCA2 mutation to be present in 11 of them (91.6%) - the highest occurrence so far reported in the literature. The CAG repeat of the affected alleles varied between 34 and 44 repeats. Age at onset and repeat length revealed an inverse correlation. Mean age at onset was 37.32 +/- 16. 74 years, and occurred earlier in males than in females. There were no differences in mean CAG repeat units between the sexes. However, a higher instability of CAG repeats was observed for paternal transmission than for maternal transmission. Age at onset and anticipation were not related to parental transmission. Our data suggest that in SCA2 an unknown sex-linked factor may play a role in the modulation of toxic effects of the polyglutamine tract.

Rapid touchdown PCR assay for the molecular diagnosis of spinocerebellar ataxia type 2.

Seven different chromosomal loci, designated SCA1 to SCA7 (spinocerebellar ataxias), have been identified as responsible for autosomal dominant cerebellar ataxias. Five genes (SCA1, 2, 3, 6, 7) have been cloned to date and show a single type of mutation, an unstable expansion of a CAG repeat coding for a polyglutamine stretch in the corresponding protein. We describe an improved polymerase chain reaction assay, based on a touchdown protocol, for the diagnosis of spinocerebellar ataxia type 2. This method produces an efficient amplification of both normal and pathological alleles and no radioactive labelling is necessary to observe the amplification products. The pathological alleles are identified by a simple non-denaturing polyacrylamide electrophoretic separation followed by ethidium bromide staining. A comparison of this technique with previously reported methods confirmed its utility for the rapid molecular diagnosis of spinocerebellar ataxia type 2. We found that the spinocerebellar ataxia type 2 mutation is responsible for 88% of the examined autosomal dominant cerebellar ataxia type 1 families in our territory (eastern Sicily). With the rapid touchdown polymerase chain reaction method, the trinucleotide expansion was also observed in 2 ataxic patients without family history of the disease, suggesting the necessity for analysis of spinocerebellar ataxia type 2 expansion even in sporadic patients.

Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons.

The connexins are the protein subunits of the gap junction intercellular channels. In the present study a new rat connexin was cloned by degenerate reverse transcription-polymerase chain reaction and its gene isolated from a mouse genomic library. The nucleotide sequence encodes a protein of 321 amino acids (called Cx36) with highly significant homology to the members of the connexin family. In situ hybridization analysis of rat brain and retina showed the strongest expression in neurons of the inferior olive, the olfactory bulb, the CA3/CA4 hippocampal subfields and several brain-stem nuclei. An intense expression was also found in the pineal gland and in the retinal ganglion cell and inner nuclear layers. Experiments with neurotoxins, locally injected in the hippocampus or specifically acting on inferior olivary neurons, confirmed the neuronal localization of Cx36. It is the first connexin to be expressed predominantly in mammalian neurons and its identification paves the way for a molecular approach in the study of the role played by gap junctions in the physiology and the pathology of the mammalian brain.

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas.

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mGluRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcript were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, in mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.