Loredana Lorusso

Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons

F3/contactin (CNTN1) and TAG-1 (CNTN2) are closely related axonal glycoproteins that are differentially regulated during development. In the cerebellar cortex TAG-1 is expressed first as granule cell progenitors differentiate in the premigratory zone of the external germinal layer. However, as these cells begin radial migration, TAG-1 is replaced by F3/contactin. To address the significance of this differential regulation, we have generated transgenic mice in which F3/contactin expression is driven by TAG-1 gene regulatory sequences, which results in premature expression of F3/contactin in granule cells. These animals (TAG/F3 mice) display a developmentally regulated cerebellar phenotype in which the size of the cerebellum is markedly reduced during the first two postnatal weeks but subsequently recovers. This is due in part to a reduction in the number of granule cells, most evident in the external germinal layer at postnatal day 3 and in the inner granular layer between postnatal days 8 and 11. The reduction in granule cell number is accompanied by a decrease in precursor granule cell proliferation at postnatal day 3, followed by an increase in the number of cycling cells at postnatal day 8. In the same developmental window the size of the molecular layer is markedly reduced and Purkinje cell dendrites fail to elaborate normally. These data are consistent with a model in which deployment of F3/contactin on granule cells affects proliferation and differentiation of these neurons as well as the differentiation of their synaptic partners, the Purkinje cells. Together, these findings indicate that precise spatio-temporal regulation of TAG-1 and F3/contactin expression is critical for normal cerebellar morphogenesis.

Treatment of spontaneously hypertensive rats with rosiglitazone ameliorates cardiovascular pathophysiology via antioxidant mechanisms in the vasculature

Oxidative stress contributes to cardiovascular complications of diabetes, in part, by reducing the bioavailability of nitric oxide (NO). We investigated the mechanisms whereby the insulin sensitizer rosiglitazone may ameliorate oxidative stress in the vasculature of spontaneously hypertensive rats (SHR). Nine-week-old SHR were treated by gavage for 7 wk with rosiglitazone (5 mg x kg(-1) x day(-1)) or vehicle control. Treatment of SHR with rosiglitazone lowered systolic blood pressure, reduced fasting plasma insulin and asymmetrical dimethylarginine, and increased insulin sensitivity (when compared with vehicle treatment). In vessel homogenates and serum from rosiglitazone-treated SHR, SOD activity was enhanced, while 8-iso-PGF(2alpha) (lipid peroxidation product) was reduced (when compared with samples from vehicle-treated SHR). Moreover, expression of p22phox (catalytic subunit of NADPH oxidase) as well as nitrotyrosine and superoxide content were all reduced in the aortas of rosiglitazone-treated SHR. In mesenteric vascular beds (MVB) isolated ex vivo from rosiglitazone-treated SHR, NO-dependent vasodilator actions of insulin were improved when compared with MVB from vehicle-treated SHR. Acute pretreatment of MVB from vehicle-treated SHR with apocynin (NADPH oxidase inhibitor) enhanced vasodilator actions of insulin (results comparable to those in MVB from rosiglitazone-treated SHR). In Langendorff heart preparations from rosiglitazone-treated SHR, ischemia/reperfusion injury caused infarcts 40% smaller than in hearts from vehicle-treated SHR. Acute pretreatment of hearts from vehicle-treated SHR with apocynin produced similar results. Finally, rosiglitazone treatment of endothelial cells in primary culture reduced superoxide induced by insulin-resistant conditions. We conclude that rosiglitazone therapy in SHR increases SOD activity and decreases p22phox expression in the vasculature to reduce oxidant stress leading to an improved cardiovascular phenotype.

Glutamic acid decarboxylase and GABA immunoreactivities in the cerebellar cortex of adult rat after prenatal exposure to a low concentration of carbon monoxide.

Glutamic acid decarboxylase and GABA immunoreactivities were qualitatively and quantitatively evaluated in the cerebellar cortex of adult rats prenatally exposed to a low concentration of carbon monoxide (75 parts per million). Carbon monoxide-exposed and control rats were perfused with modified Bouins fluid and their cerebella were embedded in paraffin. Sections from the vermis of each cerebellum were stained with Toluidine Blue or assayed with anti-glutamic acid decarboxylase 65/67 or with anti-GABA antisera. In the Toluidine Blue-stained sections, no differences were observed in the microscopic structure of the cerebellar cortex between carbon monoxide-exposed rats and controls. The distribution patterns of glutamic acid decarboxylase and GABA immunoreactivities in the cerebellar cortex of the treated animals were qualitatively comparable to those of the controls, and in accordance with previous descriptions of glutamic acid decarboxylase and GABA immunoreactivities in the rat cerebellar cortex. However, quantitative analyses demonstrated a significant reduction of immunoreactivities to both substances in the exposed rats in comparison with the controls. The reduction regarded: in the molecular layer, the number of glutamic acid decarboxylase/GABA-immunoreactive neuronal bodies and of axon terminals and the area they covered; in the Purkinje neuron layer, the number and the area covered by glutamic acid decarboxylase/GABA immunoreactive axon terminals. The differences detected in the prenatally exposed adult rats could be due to carbon monoxide-induced impairment of the differentiation of cerebellar GABA synthesizing neurons. A consequently diminished synthesis of GABA might account for some behavioral disorders detected in adult rats submitted to the same experimental procedure.

Glutamic acid decarboxylase immunoreactive large neuron types in the granular layer of the human cerebellar cortex

‘Non-traditional’ large neurons of the granular layer of the cerebellar cortex include all its large neuronal types, except the Golgi neuron, which is instead one of the five ‘classic’ types of corticocerebellar neurons. The morphological, chemical and functional characteristics of the ‘non-traditional’ large neurons have not been entirely ascertained. The aim of this study was to ascertain whether morphological evidence can be provided of GABA synthesis within the ‘non-traditional’ large neurons of the human cerebellar cortex by means of immunocytochemistry for glutamic acid decarboxylase (GAD). Fragments of postmortem cerebellar cortex of various lobules from the hemispheres and vermis were studied. Immunoreactions revealed large neurons distributed throughout the granular layer in all lobules examined. They were discriminated by analyzing the morphological features of their bodies and processes and were identified as Golgi neurons and as some ‘non-traditional’ types, such as the candelabrum, Lugaro and synarmotic neurons. In addition, immunoreactive large neurons, with their bodies and processes closely adjacent to microvessels, were observed throughout the layer: these perivascular neurons could represent a new type of ‘non-traditional’ neuron of the cerebellar cortex. This study supplies the first indication that in the human cerebellar cortex some types of ‘non-traditional’ large neurons are GAD-immunoreactive, in addition to those neurons already known to be GABAergic (i.e., stellate, basket, Purkinje and Golgi neurons). These morphological data further point out possible functional roles for GABA as a neurotransmitter/neuromodulator in intrinsic, associative and projective circuits of the cerebellar cortex.

VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex

BackgroundThe aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1.ResultsThe examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons.ConclusionThe present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.

Transgenic mice expressing F3/contactin from the transient axonal glycoprotein promoter undergo developmentally regulated deficits of the cerebellar function

We have shown that transgenic transient axonal glycoprotein (TAG)/F3 mice, in which the mouse axonal glycoprotein F3/contactin was misexpressed from a regulatory region of the gene encoding the transient axonal glycoprotein TAG-1, exhibit a transient disruption of cerebellar granule and Purkinje cell development [Development 130 (2003) 29]. In the present study we explore the neurobehavioural consequences of this mutation. We report on assays of reproductive parameters (gestation length, litter size and offspring viability) and on somatic and neurobehavioural end-points (sensorimotor development, homing performance, motor activity, motor coordination and motor learning). Compared with wild-type littermates, TAG/F3 mice display delayed sensorimotor development, reduced exploratory activity and impaired motor activity, motor coordination and motor learning. The latter parameters, in particular, were affected also in adult mice, despite the apparent recovery of cerebellar morphology, suggesting that subtle changes of neuronal circuitry persist in these animals after development is complete. These behavioural deficits indicate that the finely coordinated expression of immunoglobulin-like cell adhesion molecules such as TAG-1 and F3/contactin is of key relevance to the functional, as well as morphological maturation of the cerebellum.

Effects of prenatal exposure to the CB-1 receptor agonist WIN 55212-2 or CO on the GABAergic neuronal systems of rat cerebellar cortex

The aim of this study was to assess the effects of prenatal exposures to cannabinoids or carbon monoxide (CO) in an animal experimental model reproducing the environmental conditions in which a fetus develops whose mother, during pregnancy, ingests by smoking low doses of cannabinoids or CO. Particular attention was devoted to analyses of the long-term effects of the exposures at the level of the cerebellar cortex, where already during prenatal development the GABAergic neuronal systems may be modulated by both cannabinoids and CO. Three groups of rats were subjected to the following experimental conditions: exposure to cannabinoids by maternal treatment during pregnancy with the cannabinoid CB-1 receptor agonist WIN 55212-2 (WIN) (0.5 mg/kg/day, s.c.); exposure to CO by maternal exposure during pregnancy to CO (75 parts per million, by inhalation); and exposure to WIN+CO at the above doses and means of administration; a fourth group was used as control. The body weight of dams, length of pregnancy, litter size at birth, body weight and postnatal mortality of pups were monitored in order to evaluate possible effects of the exposures on reproduction and on prenatal and postnatal development. In the different groups, the long-term effects of the exposures were studied in adult rats (120-150 days) by light microscopy analyses of the structure of the cerebellar cortex and of the distribution in the cortex of markers of GABAergic neurons, such as GAD and GABA itself. Results. Exposures to WIN or CO did not affect reproduction or prenatal/postnatal development. Moreover, the exposed rats showed no structural alterations of the cerebellar cortex and displayed qualitative distribution patterns of GAD and GABA immunoreactivities similar to those of the controls. However, quantitative analyses indicated significant changes of both of these immunoreactivities: in comparison with the controls, they were significantly increased in WIN-exposed rats and reduced in CO-exposed rats, but not significantly different in WIN+CO-exposed rats. The changes were detected in the molecular and Purkinje neuron layers, but not in the granular layer. Prenatal exposures of rats to WIN or CO, at doses that do not affect reproduction, general processes of development and histomorphogenesis of the cerebellar cortex, cause significant changes of GAD and GABA immunoreactivities in some GABAergic neuronal systems of the adult rat cerebellar cortex, indicating selective up-regulation of GABA-mediated neurotransmission as a long-term consequence of chronic prenatal exposures to cannabinoids or CO. Because the changes consist of overexpression or, vice versa, underexpression of these immunoreactivities, functional alterations of opposite types in the GABAergic systems of the cerebellum following exposure to WIN or CO can be postulated, in agreement with the results of behavioral and clinical studies. No changes in immunoreactivities were detected after prenatal exposure to WIN and CO in association.

Chronic inflammation enhances NGF-β/TrkA system expression via EGFR/MEK/ERK pathway activation in Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune exocrine disease associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lachrymal glands). To investigate the potential implication of nerve growth factor-β (NGF-β) and its high affinity receptor tyrosine kinase receptor A (TrkA) in the regulation of pSS inflammatory responses, we studied their expression in the human salivary gland epithelial cells (SGEC) cultures from pSS minor salivary glands (MSG) biopsies and their relationship with histopathological disease parameters. Here, we demonstrated an increased expression of the NGF-β/TrkA system in pSS SGEC, correlated with the MSG inflammation grade. The results demonstrate that the pro-inflammatory cytokines TNF-α and IL-6 enhance NGF-β production; on the contrary, NGF-β production was reduced in the presence of both Raf-1 kinase and MEK inhibitors. Furthermore, TNF-α/IL-6 treatment increased ERK1/2 phosphorylation. Inhibition of the EGF/EGFR system also decreased NGF-β release by pSS SGEC, indicating that the chronic inflammatory condition characteristic of pSS enhances NGF-β production via EGFR/Raf-1/MEK/ERK pathway activation.Key messageNGF-β and TrkA expression is elevated in salivary gland epithelial cells of primary Sjögren’s syndrome (pSS).Overexpression of NGF-β/TrkA system in pSS occurs via EGFR/Raf-1/MEK/ERK pathway.In pSS, NGF-β overexpression was prevented by EGFR/Raf-1/MEK/ERK pathway inhibition.

Interleukin-15 as a potential new target in Sjögren's syndrome-associated inflammation.

IL-15 is a key regulatory cytokine that shares many biological properties with IL-2. Recently, it has been shown that IL-15 could be up-regulated in T cell-mediated inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases. However, the role and expression of IL-15 in the inflammatory autoimmune disease Sjögrens syndrome (SS) has not been investigated. In the present study we evaluated the expression of IL-15 mRNA and protein in minor salivary gland (MSG) biopsy specimens and in human salivary gland epithelial cell (SGEC) cultures obtained from patients with primary SS (pSS) and compared their expression with that seen in normal healthy control subjects. IL-15 gene and protein analysis revealed that SGEC are able to produce IL-15. Results obtained demonstrated that the number of IL-15(+) cultured SGEC was significantly higher in cells derived from patients with pSS in comparison with SGEC from healthy subjects; similar results were obtained for IL-15 immunoreactivity by using immunohistochemistry that revealed a strong expression both in acinar and in ductal cells from pSS MSG. These studies could provide a rational basis to determine whether IL-15 could be a good candidate for anti-cytokine therapy in chronic inflammatory pSS diseases.

Calbindin‐D28K Immunoreactivity in the Human Cerebellar Cortex

Calbindin‐D28k (CB) is a calcium‐binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer‐specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed. Anat Rec, 297:1306–1315, 2014.