Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis.

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

The expression of ceruloplasmin in astrocytes is essential for postnatal myelination and myelin maintenance in the adult brain.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux. The absence of this protein in humans and rodents produces progressive neurodegeneration with brain iron accumulation. Astrocytes express high levels of Cp and iron efflux from these cells has been shown to be central for oligodendrocyte maturation and myelination. To explore the role of astrocytic Cp in brain development and aging we generated a specific conditional KO mouse for Cp in astrocytes (Cp cKO). Deletion of Cp in astrocytes during the first postnatal week induced hypomyelination and a significant delay in oligodendrocyte maturation. This abnormal myelin synthesis was exacerbated throughout the first two postnatal months and accompanied by a reduction in oligodendrocyte iron content, as well as an increase in brain oxidative stress. In contrast to young animals, deletion of astrocytic Cp at 8 months of age engendered iron accumulation in several brain areas and neurodegeneration in cortical regions. Aged Cp cKO mice also showed myelin loss and oxidative stress in oligodendrocytes and neurons, and at 18 months of age, developed abnormal behavioral profiles, including deficits in locomotion and short-term memory. In summary, our results demonstrate that iron efflux-mediated by astrocytic Cp-is essential for both early oligodendrocyte maturation and myelin integrity in the mature brain. Additionally, our data suggest that astrocytic Cp activity is central to prevent iron accumulation and iron-induced oxidative stress in the aging CNS.

Ceruloplasmin deletion in myelinating glial cells induces myelin disruption and oxidative stress in the central and peripheral nervous systems.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux and has been postulated to have a neuroprotective role. During the myelination process, oligodendrocytes (OLs) and Schwann cells (SCs) express high levels of Cp, but the role of this enzyme in glial cell development and function is completely unknown. To define the function of Cp in the myelination of the central and peripheral nervous systems, we have conditionally knocked-out Cp specifically in OLs and SCs during early postnatal development as well as in aged mice. Cp ablation in early OLs (postnatal day 2, P2) significantly affects the differentiation of these cells and the synthesis of myelin through the first four postnatal weeks. The total number of mature myelinating OLs was reduced, and the density of apoptotic OLs was increased. These changes were accompanied with reductions in the percentage of myelinated axons and increases in the g-ratio of myelinated fibers. Cp ablation in young myelinating OLs (P30 or P60) did not affect myelin synthesis and/or OL numbers, however, Cp loss in aged OLs (8 months) induced cell iron overload, apoptotic cell death, brain oxidative stress, neurodegeneration and myelin disruption. Furthermore, Cp deletion in SCs affected postnatal SC development and myelination and produced motor coordination deficits as well as oxidative stress in young and aged peripheral nerves. Together, our data indicate that Cp ferroxidase activity is essential for OLs and SCs maturation during early postnatal development and iron homeostasis in matured myelinating cells. Additionally, our results suggest that Cp expression in myelinating glial cells is crucial to prevent oxidative stress and neurodegeneration in the central and peripheral nervous systems.

Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

Voltage-gated Ca2+ entry promotes oligodendrocyte progenitor cell maturation and myelination in vitro.

We have previously shown that the expression of voltage-operated Ca(++) channels (VOCCs) is highly regulated in the oligodendroglial lineage and is essential for proper oligodendrocyte progenitor cell (OPC) migration. Here we assessed the role of VOCCs, in particular the L-type, in oligodendrocyte maturation. We used pharmacological treatments to activate or block voltage-gated Ca(++) uptake and siRNAs to specifically knock down the L-type VOCC in primary cultures of mouse OPCs. Activation of VOCCs by plasma membrane depolarization increased OPC morphological differentiation as well as the expression of mature oligodendrocyte markers. On the contrary, inhibition of L-type Ca(++) channels significantly delayed OPC development. OPCs transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents showed reduce Ca(++) influx by ~75% after plasma membrane depolarization, indicating that Cav1.2 is heavily involved in mediating voltage-operated Ca(++) entry in OPCs. Cav1.2 knockdown induced a decrease in the proportion of oligodendrocytes that expressed myelin proteins, and an increase in cells that retained immature oligodendrocyte markers. Moreover, OPC proliferation, but not cell viability, was negatively affected after L-type Ca(++) channel knockdown. Additionally, we have tested the ability of L-type VOCCs to facilitate axon-glial interaction during the first steps of myelin formation using an in vitro co-culture system of OPCs with cortical neurons. Unlike control OPCs, Cav1.2 deficient oligodendrocytes displayed a simple morphology, low levels of myelin proteins expression and appeared to be less capable of establishing contacts with neurites and axons. Together, this set of in vitro experiments characterizes the involvement of L-type VOCCs on OPC maturation as well as the role played by these Ca(++) channels during the early phases of myelination.

The dysbindin-containing complex (BLOC-1) in brain: developmental regulation, interaction with SNARE proteins and role in neurite outgrowth.

Previous studies have implicated DTNBP1 as a schizophrenia susceptibility gene and its encoded protein, dysbindin, as a potential regulator of synaptic vesicle physiology. In this study, we found that endogenous levels of the dysbindin protein in the mouse brain are developmentally regulated, with higher levels observed during embryonic and early postnatal ages than in young adulthood. We obtained biochemical evidence indicating that the bulk of dysbindin from brain exists as a stable component of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a multi-subunit protein complex involved in intracellular membrane trafficking and organelle biogenesis. Selective biochemical interaction between brain BLOC-1 and a few members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) superfamily of proteins that control membrane fusion, including SNAP-25 and syntaxin 13, was demonstrated. Furthermore, primary hippocampal neurons deficient in BLOC-1 displayed neurite outgrowth defects. Taken together, these observations suggest a novel role for the dysbindin-containing complex, BLOC-1, in neurodevelopment, and provide a framework for considering potential effects of allelic variants in DTNBP1--or in other genes encoding BLOC-1 subunits--in the context of the developmental model of schizophrenia pathogenesis.

Knocking-down the NMDAR1 subunit in a limited amount of neurons in the rat hippocampus impairs learning.

Amplicon vectors derived from herpes simplex virus type 1 were built to modify NMDA receptors by expressing antisense RNA for the essential NR1 subunit. Their ability to modify endogenous levels of NR1 was tested in cultures of rat embryo neocortical neurons. We studied behaviour and tested for expression in adult rats injected with those vectors into the dorsal hippocampus to find out which cells and how many appear involved in memory formation. Rats injected with vectors expressing NR1 antisense performed significantly worse than control rats in an inhibitory avoidance task. Immunohistochemistry was performed in brain slices from the same animals. The transduced cells represented 6-7% of pyramidal neurons in CA1, showing that a single gene knockdown of NR1 in a small number of neurons significantly impaired memory formation. Perhaps neurons undergoing synaptic plasticity are more susceptible to NR1 knockdown, and hence NMDAR are particularly required in those neurons undergoing synaptic plasticity during learning, or perhaps, and more likely, there is not a high level of redundancy in the hippocampal circuits involved, leading to the idea that a certain level of NR1 expression/availability appears necessary for memory formation in most of CA1 pyramidal neurons.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.

Is type 2 diabetes a different disease in obese and nonobese patients?

OBJECTIVE: The main purpose of this work was to study the possible differences in insulin secretion in a large group of type 2 diabetic patients in relation to diabetes duration, obesity, and the presence of secondary failure after treatment with oral hypoglycemic agents. RESEARCH DESIGN AND METHODS: There were 147 nonobese and 215 obese type 2 diabetic subjects, aged 35-80 years, investigated in a cross-sectional descriptive study Subjects were grouped according to whether glycemic control was good (mean blood glucose <8.5 mmol/l) or poor. Beta-cell function was assessed by measuring meal-stimulated insulin and C-peptide concentrations, as the mean of the three postprandial increments above the premeal value. RESULTS: Basal C-peptide concentrations were significantly higher in obese than nonobese patients of both groups. The mean of meal-stimulated C-peptide concentrations was also significantly higher in obese than nonobese patients with good glycemic control, but not in the secondary failure groups. In nonobese and obese patients considered separately, a significant negative correlation between the mean of daily blood glucose and meal-stimulated C-peptide was observed (r=-0.705 and r=-0.679, respectively, P < 0.001) and the residual beta-cell function was significantly correlated with the known duration of diabetes and metabolic control, but not with BMI, in both groups. CONCLUSIONS: On average, obese diabetic subjects showed higher meal-stimulated C-peptide than nonobese subjects only in well-controlled groups. In both obese and nonobese patients, an inverse association between meal-stimulated insulin secretion and duration of diabetes was observed. In obese patients, as in nonobese patients, the lower beta-cell function seems likely to be the major pathogenetic factor in the appearance of secondary failure, while being overweight plays only a minor role, thus showing that type 2 diabetes is the same disease in obese and nonobese patients.

Progressive deterioration of beta-cell function in nonobese type 2 diabetic subjects. Postprandial plasma C-peptide level is an indication of insulin dependency.

The purpose of the present study was to characterize secondary failure (SF) to oral hypoglycaemic agents by assessment of threshold insulin-secretion values in relation to diabetes duration. One hundred and forty-seven nonobese diabetic patients, 35 to 80 years of age, with disease duration ranging from 1 to 36 years, were studied. Beta-cell function was assessed by meal-stimulated (delta CP) and glucagon-stimulated (delta aCP) C-peptide concentrations. The quality of glycaemic control was considered good if mean daily blood glucose was less than 8.5 mmol/l. One group with good (NOb-GC) and another with poor control (NOb-SF) were established. Mean daily glycaemia was negatively correlated with delta CP or delta aCP (r = -0.703 vs r = -0.696; p < 0.001) more than with basal C-peptide (r = -0.453; p < 0.001). A close positive correlation between meal-stimulated (delta CP) and glucagon-stimulated (delta aCP) C-peptide concentrations was observed (r = 0.869; p < 0.001). Residual beta-cell function (delta CP and delta aCP) was significantly correlated with known disease duration in both groups (GC: r = -0.693 and SF: r = -0.680; p < 0.001). Nonobese patients with SF showed early impaired secretion during the first years of disease, meal-stimulated delta CP being below 0.350 mmol/l. The most useful result in this study was the incremental value of C-peptide (delta CP), which showed minimal overlapping between the two groups. Basal, postprandial or postglucagon absolute values were less discriminating. The daily profile allowed measurement of both glycaemic control and insulin production after a regular meal. The validity of this measurement was confirmed by the strong correlation between meal-stimulated and glucagon-stimulated delta C-peptide concentrations. This parameter is a useful physiological marker of secondary failure.

[The utility of postprandial C-peptide evaluation in type 2 diabetes]. / Utilità del C-peptide postprandiale nella valutazione del diabete tipo 2.

The secondary drug failure is a well known phenomenon in the development of type 2 diabetes mellitus, but an exact definition of this situation is still lacking. The aim of this research was to evaluate the beta-cell reserve in non obese diabetic patients in relation to the metabolic control and the duration of disease. The main aim was to identify values of postprandial plasma C-peptide that can characterize the patients requiring insulin treatment. A daily profile was performed in 135 non obese diabetic patients, within 20% of their ideal body weight. The mean diurnal values (m) and the mean post-prandial increases (delta mpp) of plasma glucose (G), insulin (IRI) and C-peptide (CP) were assessed. Fourty-four patients showed good (NwD-GC: G-m = 138 +/- 3.2 mg/dl) and 91 poor metabolic control (NwD-SF: G-m = 210 +/- 4.8 mg/dl), according to the G-m lower or higher than 150 mg/dl. Beta-cell reserve (CP-delta mpp: 0.70 +/- 0.03 vs 1.39 +/- 0.04 ng/ml) and C-peptide/insulin molar ratio (CP/IRA-delta mpp: 2.36 +/- 0.06 vs 2.80 +/- 0.06) were significantly lower (p < 0.001) in NwD-SF than in NwD-GC. NwD-GC and NwD-SF were respectively divided into three subgroups, according to the duration of disease. A progressive reduction of CP-delta mpp and an increase in SF prevalence, from the first to the third decade of diabetes duration, was observed. In both NwD-Gc and NwD-SF the duration of disease inversely correlated with CP-delta mpp (NwD-GC: y = 1.59-0.019X, p < 0.001; NwD-SF: y = 1.01-0.023X, p < 0.001). The analysis of the two regression lines showed that patients with CP-delta values lower than 1.0 ng/ml require insulin treatment. In conclusion the duration of diabetes and the progressive reduction of beta-cell reserve represent the major pathogenetic factors in secondary failure.

Prevalence of micro- and macroalbuminuria and their relationship with other cardiovascular risk factors in essential hypertension.

A cohort of 227 untreated essential hypertensive patients from north-western Italy was studied in order to evaluate the prevalence of micro- and macroalbuminuria and their relationship with other cardiovascular risk factors. Albuminuria was evaluated as the albumin to creatinine ratio (Alb/Cr) in three non-consecutive first morning samples. The prevalence of microalbuminuria and macroalbuminuria was 10% and 2.2%, respectively. Albuminuric patients showed higher blood pressure, serum creatinine, triglycerides and uric acid as well as a greater prevalence of retinopathy. Stepwise multiple regression analysis demonstrated that only a small part of variations in albuminuria was explained by changes in blood pressure. Duration of disease did not seem to influence microalbuminuria. The presence of hypertensive retinopathy was associated with greater albuminuria, longer duration of hypertension, and higher prevalence of major ECG changes, but not with higher blood pressure levels. Microalbuminuria, rather than a consequence of elevated blood pressure levels, seems to be a marker of a syndrome featuring, among other characteristics, essential hypertension. Furthermore, microalbuminuria must be considered as an independent cardiovascular risk factor.

[Multiple symmetrical lipomatosis. Metabolic effects of excision of lipomatous tissue]. / Lipomatosi multipla simmetrica. Effetti metabolici dell'exeresi di tessuto lipomatoso.

Multiple Symmetric Lipomatosis (MSL) is a syndrome characterized by the occurrence of symmetric lipomas over various regions of the body. No clear etiology has been recognized while a frequent association with systemic metabolic abnormalities has been described. The metabolic situation of a subject affected by MSL was assessed before and after surgical excision of lipomas. A condition of impaired glucose tolerance (IGT) was verified both before and after surgery by the performance of oral glucose tolerance test, glucagon test, and daily glucose profile. No significant differences were observed after the ablation of lipomatous masses with regard to glucose, IRI, IRCP and NEFA behaviour. We concluded that the resection of lipomas can not modify glucose tolerance in MSL and that lipomas can be considered as tissues metabolically independent from the rest of body fat.

Blood lactate behavior after glucose load in diabetes mellitus.

The aim of this study was to evaluate the relationships between blood lactate and plasma glucose, insulin (IRI) and C-peptide (IRCP) during the first hour of an oral glucose load (OGTT, 100 g). Twelve controls, sixteen non-insulin-dependent (NIDDM) and four insulin-dependent (IDDM) diabetic subjects were studied. A significant increase in blood lactate was observed at 15 min in normal subjects, whereas there was a delayed increase at 45 min in NIDDM subjects, in the presence of IRCP increments of 0.31 nmol/l. In order to have a minimum significant lactate increment, the threshold value of peripheral IRCP increment was about 0.30 nmol/l. In IDDM subjects, despite considerable hyperglycemia, blood lactate concentration remained unchanged throughout the test. In normal and NIDDM subjects there was a significant negative correlation between delta lactate and delta glucose (r = -0.89, p less than 0.001) and a significant positive correlation between delta lactate and delta IRCP (r = 0.78, p less than 0.001). In conclusion, hyperglycemia itself and the lack of increase in insulin secretion do not affect blood lactate increase during OGTT; blood concentration of this metabolite depends mainly on an early insulin secretion apt to enhance tissue glucose uptake and to inhibit gluconeogenesis.