Epigenetic reprogramming of cortical neurons through alteration of dopaminergic circuits.

Alterations of the dopaminergic system are associated with the cognitive and functional dysfunctions that characterize complex neuropsychiatric disorders. We modeled a dysfunctional dopaminergic system using mice with targeted ablation of dopamine (DA) D2 autoreceptors in mesencephalic dopaminergic neurons. Loss of D2 autoreceptors abolishes D2-mediated control of DA synthesis and release. Here, we show that this mutation leads to a profound alteration of the genomic landscape of neurons receiving dopaminergic afferents at distal sites, specifically in the prefrontal cortex. Indeed, we observed a remarkable downregulation of gene expression in this area of ~2000 genes, which involves a widespread increase in the histone repressive mark H3K9me2/3. This reprogramming process is coupled to psychotic-like behaviors in the mutant mice. Importantly, chronic treatment with a DA agonist can revert the genomic phenotype. Thus, cortical neurons undergo a profound epigenetic reprogramming in response to dysfunctional D2 autoreceptor signaling leading to altered DA levels, a process that may underlie a number of neuropsychiatric disorders.

Outer retina dysfunction and choriocapillaris impairment in type 1 diabetes.

To study the outer retina morpho-functional characteristics and the choriocapillaris (CC) features in type 1 diabetic (T1D) patients, with and without signs of diabetic retinopathy (NPDR and NoDR). Twenty-five NPDR and 18 NoDR eyes were imaged by Optical Coherence Tomography Angiography. Ellipsoid zone (EZ) "normalized" reflectivity and CC perfusion density parameters, as flow deficits number (FDn), flow deficit average area (FDa) and flow deficit percentage (FD%), were analysed. Multifocal electroretinogram (mfERG) response amplitude densities (RADs) were measured. Mean EZ "normalized" reflectivity, CC FDn and FD% values, were similar (p > 0.05) in both groups, FDa was significant greater (p > 0.05) in NPDR compared with NoDR eyes. MfERG-RADs were similar in both groups. NPDR eyes showed a significant (p < 0.05) linear correlation between RADs and both, CC FDa and FD%. The EZ "normalized" reflectivity was negatively correlated with CC FD% in NoDR eyes. In NPDR T1D eyes a significant relationship between abnormal outer retina functional responses and CC impairment was observed, while in NoDR eyes the photoreceptor reflectivity was correlated to CC abnormalities. The outer retina dysfunction in NPDR correlated to CC drop-out let hypothesize that the outer retinal elements are functionally impaired in proportion to the CC vascular supply deficit.

A transition clinic model for inflammatory bowel disease between two tertiary care centers: outcomes and predictive factors.

OBJECTIVE: Few models of transition have been proposed for inflammatory bowel disease (IBD). The aim of the present study is to evaluate the feasibility of a transition model and the predictive factors for success/failure. PATIENTS AND METHODS: Patients with low activity or remission IBD were enrolled. Proposed model: three meetings every four-six weeks: the first one in the pediatric center (Bambino Gesù Children's Hospital); the second one, in the adult center (Foundation Polyclinic University A. Gemelli), with pediatric gastroenterologists; the last one, in the adult center, with adult gastroenterologists only. Questionnaires included anxiety and depression clinical scale, self-efficacy, quality of life, visual-analogic scale (VAS). Transition was considered successful if the three steps were completed. RESULTS: Twenty patients were enrolled (range 18-25 years; M/F: 12/8; Ulcerative Colitis/Crohn's Disease 10/10); eight accepted the transition program, four delayed the process and eight refused. Patients who completed transition generated higher scores on the resilience scale, better scores on well-being perception, and had lower anxiety scores. Patients who failed transition were mostly women. The perceived utility of the transition program was scored 7.3 on a VAS scale. CONCLUSIONS: The proposed transition program seems to be feasible. Psychological scores may help in selecting patients and predicting outcomes.

Targeting of G alpha i2 to the Golgi by alternative spliced carboxyl-terminal region.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) may participate in membrane traffic events. A complementary DNA (cDNA) was isolated from a mouse pituitary cDNA library that corresponded to an alternatively spliced form of the gene encoding the G protein alpha subunit G alpha i2. The cDNA was identical to that encoding G alpha i2 except that the region encoding for the carboxyl-terminal 24 amino acids was replaced by a longer region encoding 35 amino acids that have no sequence similarity with G alpha i2 or other members of the G protein family. This alternative spliced product and the corresponding protein (sGi2) were present in several tissues. Specific antibodies revealed that sGi2 was localized in the Golgi apparatus, suggesting a role in membrane transport. Thus, alternative splicing may generate from a single gene two G protein alpha subunits with differential cellular localization and function.

Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products.

The products of the adenovirus-2 (Ad2) immortalizing oncogene E1A repress the activity of the SV40, polyoma virus and E1A enhancers. Evidence is presented that Ad2 infection of MPC11 plasmocytoma cells results in an inhibition of transcription of both the gamma 2b heavy chain (IgH) and the kappa light chain immunoglobulin genes. This inhibition is caused by the Ad2 E1A products. Furthermore, the Ad2 E1A products repress transcription activated by the immunoglobulin heavy chain enhancer in chimeric recombinants, which are either stably integrated in the genome of lymphoid cells or are present as episomes. The implications of negative regulation of cellular enhancers are discussed.

Impaired locomotion and dopamine signaling in retinoid receptor mutant mice.

In the adult mouse, single and compound null mutations in the genes for retinoic acid receptor beta and retinoid X receptors beta and gamma resulted in locomotor defects related to dysfunction of the mesolimbic dopamine signaling pathway. Expression of the D1 and D2 receptors for dopamine was reduced in the ventral striatum of mutant mice, and the response of double null mutant mice to cocaine, which affects dopamine signaling in the mesolimbic system, was blunted. Thus, retinoid receptors are involved in the regulation of brain functions, and retinoic acid signaling defects may contribute to pathologies such as Parkinson's disease and schizophrenia.

Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia.

The function of dopamine (DA) in the nervous system is paralleled by its neuroendocrine control of pituitary gland functions. Here, we document the neuroendocrine function of dopamine by studying the pituitary gland of mice lacking DA D2 receptors (D2R). These mice present a striking, progressive increase in lactotroph number, which ultimately leads to tumors in aged animals. Females develop tumors much earlier than males. An estrogen-mediated lactotroph proliferation cannot account for this sexual dimorphism, since D2R-null females are hypoestrogenic and, thus, have estrogen levels similar to males. In contrast, prolactin levels are six times higher in females than in males. We show that active prolactin receptors are present in the pituitary and their expression increases in concomitance with tumor expansion. These results point to prolactin as an autocrine proliferative factor in the pituitary gland. Additionally, they demonstrate an antiproliferative function for DA regulated through D2 receptor activation.

Mutations in signal transduction pathways and inherited diseases.

Intracellular signal transduction pathways have central roles in processes such as growth, differentiation, neurotransmission and development. The aberrant expression of components of various signal transduction pathways has profound consequences for cellular functions. Recent findings indicate that many cases of neoplasia and inherited diseases have, at their roots, mutations in key steps of signalling pathways.

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

Ozone: A Multifaceted Molecule with Unexpected Therapeutic Activity.

A comprehensive outline for understanding and recommending the therapeutic use of ozone in combination with established therapy in diseases characterized by a chronic oxidative stress is currently available. The view of the absolute ozone toxicity is incorrect, because it has been based either on lung or on studies performed in artificial environments that do not correspond to the real antioxidant capacity of body compartments. In fact, ozone exerts either a potent toxic activity or it can stimulate biological responses of vital importance, analogously to gases with prospective therapeutic value such as NO, CO, H2S, H2, as well as O2 itself. Such a crucial difference has increasingly become evident during the last decade. The purpose of this review is to explain the aspects still poorly understood, highlighting the divergent activity of ozone on the various biological districts. It will be clarified that such a dual effect does not depend only upon the final gas concentration, but also on the particular biological system where ozone acts. The real significance of ozone as adjuvant therapeutic treatment concerns severe chronic pathologies among which are cardiovascular diseases, chronic obstructive pulmonary diseases, multiple sclerosis, and the dry form of age-related macular degeneration. It is time for a full insertion of ozone therapy within pharmaceutical sciences, responding to all the requirements of quality, efficacy and safety, rather than as either an alternative or an esoteric approach.

Neuroretinal alterations in the early stages of diabetic retinopathy in patients with type 2 diabetes mellitus.

PurposeTo study neuroretinal alterations in patients affected by type 2 diabetes with no diabetic retinopathy (DR) or mild nonproliferative diabetic retinopathy (NPDR) and without any sign of diabetic macular edema.Patients and methodsIn total, 150 type 2 diabetic patients with no (131 eyes) or mild NPDR (19 eyes) and 50 healthy controls were enrolled in our study. All underwent a complete ophthalmologic examination, including Spectral-Domain optical coherence tomography (SD-OCT). Ganglion cell-inner plexiform layer (GC-IPL) and retinal nerve fiber layer (RNFL) thickness values were calculated after automated segmentation of SD-OCT scans.ResultsMean best-corrected visual acuity was 0.0±0.0 LogMAR in all the groups. Mean GC-IPL thickness was 80.6±8.1 µm in diabetic patients and 85.3±9.9 µm in healthy controls, respectively (P=0.001). Moreover, evaluating the two different diabetic groups, GC-IPL thickness was 80.7±8.1 µm and 79.7±8.8 µm in no-DR and mild-NPDR group (P=0.001 and P=0.022 compared with healthy controls, respectively). Average RNFL thickness was 86.1±10.1 µm in diabetes patients and 91.2±7.3 µm in controls, respectively (P=0.003). RNFL thickness was 86.4±10.2 µm in no-DR group and 84.1±9.4 µm in mild-NPDR group (P=0.007 and P=0.017 compared with healthy controls, respectively).ConclusionWe demonstrated a significantly reduced GC-IPL and RNFL thickness values in both no-DR and mild-NPDR groups compared with healthy controls. These data confirmed neuroretinal alterations are early in diabetes, preceding microvascular damages.

Neuroprotective role of dopamine against hippocampal cell death.

Glutamate excitotoxicity plays a key role in the induction of neuronal cell death occurring in many neuropathologies, including epilepsy. Systemic administration of the glutamatergic agonist kainic acid (KA) is a well characterized model to study epilepsy-induced brain damage. KA-evoked seizures in mice result in hippocampal cell death, with the exception of some strains that are resistant to KA excitotoxicity. Little is known about the factors that prevent epilepsy-related neurodegeneration. Here we show that dopamine has such a function through the activation of the D2 receptor (D2R). D2R gene inactivation confers susceptibility to KA excitotoxicity in two mouse strains known to be resistant to KA-induced neurodegeneration. D2R-/- mice develop seizures when administered KA doses that are not epileptogenic for wild-type (WT) littermates. The spatiotemporal pattern of c-fos and c-jun mRNA induction well correlates with the occurrence of seizures in D2R-/- mice. Moreover, KA-induced seizures result in extensive hippocampal cell death in D2R-/- but not WT mice. In KA-treated D2R-/- mice, hippocampal neurons die by apoptosis, as indicated by the presence of fragmented DNA and the induction of the proapoptotic protein BAX. These results reveal a central role of D2Rs in the inhibitory control of glutamate neurotransmission and excitotoxicity.

A transgenic mouse model for inducible and reversible dysmyelination.

Oligodendrocytes are glial cells devoted to the production of myelin sheaths. Myelination of the CNS occurs essentially after birth. To delineate both the times of oligodendrocyte proliferation and myelination, as well as to study the consequence of dysmyelination in vivo, a model of inducible dysmyelination was developed. To achieve oligodendrocyte ablation, transgenic animals were generated that express the herpes virus 1 thymidine kinase (HSV1-TK) gene under the control of the myelin basic protein (MBP) gene promoter. The expression of the MBP-TK transgene in oligodendrocytes is not toxic on its own; however, toxicity can be selectively induced by the systemic injection of animals with nucleoside analogs, such as FIAU [1-(2-deoxy-2-fluoro-beta-delta-arabinofuranosyl)-5-iodouracil]. This system allows us to control the precise duration of the toxic insult and the degree of ablation of oligodendrocytes in vivo. We show that chronic treatment of MBP-TK mice with FIAU during the first 3 postnatal weeks triggers almost a total depletion of oligodendrocytes in the CNS. These effects are accompanied by a behavioral phenotype characterized by tremors, seizures, retarded growth, and premature animal death. We identify the period of highest oligodendrocytes division in the first 9 postnatal days. Delaying the beginning of FIAU treatments results in different degrees of dysmyelination. Dysmyelination in MBP-TK mice is always accompanied by astrocytosis. Thus, this transgenic line provides a model to study the events occurring during dysmyelination of various intensities. It also represents an invaluable tool to investigate remyelination in vivo.

Inhibition of dopamine release via presynaptic D2 receptors: time course and functional characteristics in vivo.

Most neurotransmitters inhibit their own release through autoreceptors. However, the physiological functions of these presynaptic inhibitions are still poorly understood, in part because their time course and functional characteristics have not been described in vivo. Dopamine inhibits its own release through D2 autoreceptors. Here, the part played by autoinhibition in the relationship between impulse flow and dopamine release was studied in vivo in real time. Dopamine release was evoked in the striatum of anesthetized mice by electrical stimulation of the medial forebrain bundle and was continuously monitored by amperometry using carbon fiber electrodes. Control experiments performed in mice lacking D2 receptors showed no autoinhibition of dopamine release. In wild-type mice, stimulation at 100 Hz with two to six pulses linearly inhibited further release, whereas single pulses were inefficient. Dopaminergic neurons exhibit two discharge patterns: single spikes forming a tonic activity below 4 Hz and bursts of two to six action potentials at 15 Hz. Stimulation mimicking one burst (four pulses at 15 Hz) promoted extracellular dopamine accumulation and thus inhibited further dopamine release. This autoinhibition was maximal between 150 and 300 msec after stimulation and disappeared within 600 msec. This delayed and prolonged time course is not reflected in extracellular DA availability and thus probably attributable to mechanisms downstream from autoreceptor stimulation. Thus, in physiological conditions, autoinhibition has two important roles. First, it contributes to the attenuation of extracellular dopamine during bursts. Second, autoinhibition elicited by one burst transiently attenuates further dopamine release elicited by tonic activity.

The role of dopamine receptors in regulating the size of axonal arbors.

Factors that regulate terminal arbor size of substantia nigra pars compacta (SNpc) neurons during development and after injury are not well understood. This study examined the role of dopamine receptors in regulating arbor size. Terminal arbors were examined in mice with targeted deletion of the D1 or D2 dopamine receptor [D1(-/-) and D2(-/-) mice, respectively]. Terminal trees were also examined after treatment with receptor blockers and after partial SNpc lesions. Immunohistochemistry was performed, and the number of SNpc neurons and dopaminergic terminals in the striatum was estimated. The number of dopaminergic SNpc neurons were reduced in D1(-/-) and D2(-/-) mice. Density of dopaminergic terminals was unchanged in D1(-/-) mice and increased in D2 (-/-) mice. Steady-state striatal DA and DOPAC levels revealed that dopamine activity was enhanced in D2(-/-) mice but reduced in D1(-/-) mice. Two months after partial SNpc lesions, striatal terminal density was normal in both wild-type and D1(-/-) mice but reduced in D2(-/-) mice. Administration of DA receptor antagonists resulted in larger terminal arbors in D1(-/-) and wild-type mice, whereas D2(-/-) mice showed no change in terminal density. Functional blockade of the D2R during development or in the adult brain results in increased axonal sprouting. Partial SNpc lesions resulted in compensatory sprouting, only in mice with functional D2R. These results suggest that individual dopaminergic axons in D2(-/-) mice have reached maximal arbor size. We conclude that the D2 receptor may play a role in modulating the extent of the terminal arbor of SNpc neurons.

Inhibition of mitochondrial complex II induces a long-term potentiation of NMDA-mediated synaptic excitation in the striatum requiring endogenous dopamine.

Abnormal involuntary movements and cognitive impairment represent the classical clinical symptoms of Huntington's disease (HD). This genetic disorder involves degeneration of striatal spiny neurons, but not striatal large cholinergic interneurons, and corresponds to a marked decrease in the activity of mitochondrial complex II [succinate dehydrogenase (SD)] in the brains of HD patients. Here we have examined the possibility that SD inhibitors exert their toxic action by increasing glutamatergic transmission. We report that SD inhibitors such as 3-nitroproprionic acid (3-NP), but not an inhibitor of mitochondrial complex I, produce a long-term potentiation of the NMDA-mediated synaptic excitation (3-NP-LTP) in striatal spiny neurons. In contrast, these inhibitors had no effect on excitatory synaptic transmission in striatal cholinergic interneurons and pyramidal cortical neurons. 3-NP-LTP involves increased intracellular calcium and activation of the mitogen-activated protein kinase extracellular signal-regulated kinase and is critically dependent on endogenous dopamine acting via D2 receptors, whereas it is negatively regulated by D1 receptors. Thus 3-NP-LTP might play a key role in the regional and cell type-specific neuronal death observed in HD.

Absence of dopaminergic control on melanotrophs leads to Cushing's-like syndrome in mice.

Dopamine negatively regulates POMC gene expression in melanotrophs of the intermediate lobe of the pituitary gland. The dopaminergic receptor involved in this control is the dopamine D2 receptor (D2R). The principal products of the POMC gene in melanotrophs are beta-endorphin and alpha-MSH. POMC is differently processed in the corticotrophs, where it is not regulated by dopamine and it is principally processed into ACTH. Here we show that D2R-deficient mice have increased POMC expression and intermediate lobe hypertrophy. Strikingly, D2R-deficient mice have unexpected elevated ACTH levels with a corresponding increase of corticosteroids and consequent hypertrophy of the adrenal gland. This phenotype is reminiscent of Cushing's syndrome in humans. Interestingly, we show that the elevation in ACTH levels is due to an aberrant processing of POMC in melanotrophs. Indeed, we demonstrate that in addition to controlling POMC gene expression in these cells, dopamine, by modulating the expression of the convertases involved in the cleavage of the POMC prohormone, strictly regulates its processing. These results reveal a key role for dopamine in the control of POMC-derived peptides and furthermore indicate an implication of the dopaminergic system in the genesis of Cushing's syndrome.

Preferential coupling between dopamine D2 receptors and G-proteins.

The D2 dopamine receptor, an inhibitor of adenylyl cyclase, belongs to the family of seven transmembrane domain G-protein-coupled receptors. This receptor is encoded by two mRNAs produced from the same gene by alternative splicing, here referred to as D2L and D2S. The resultant proteins are identical except for an insertion of 29 amino acids in the putative third intracytoplasmic domain. This domain has been shown to be important for the coupling of this family of receptors to G-proteins. We have previously shown that there is differential inhibition of the adenylyl cyclase activity when these two receptors are produced in JEG3 cells; D2S is more efficient than D2L. To understand the molecular basis of such differential activity, we analyzed the G-proteins expressed in these cells. Here we show that G alpha i2 is absent in this cell line. Moreover, it is possible to restore the same inhibitory activity obtained by D2S when an expression vector encoding this alpha-subunit is cotransfected with D2L. In addition, transfections of the two receptors in a recipient cell line containing the three G alpha i subtypes confirm that the two receptors behave similarly. We conclude that the 29-amino acid insertion present in D2L allows it to interact specifically with G alpha i2. These data suggest that in vivo the function of activated D2 receptors is exerted by specific interactions with Gi-protein subtypes.