Stefano Govoni

Dopamine receptors: Pharmacological and anatomical evidences indicate that two distinct dopamine receptor populations are present in rat striatum

(3H)-haloperidol and (3H)-spiroperidol binding studies after kainate injection into the striatum indicate the presence of dopamine receptive sites not located on post-synaptic membranes. The majority of these dopamine “receptors” which are not associated to an adenylate cyclase appear to be localized on terminals of cortico-striatal neurons. On the other hand sulpiride, an antipsychotic drug which increases striatal dopamine synthesis, does not inhibit dopamine-stimulated adenylate cyclase either in vitro or in vivo. However sulpiride stereospecifically displaces (3H)-haloperidol from striatal binding sites.

Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway

Several lines of biochemical evidence correlate the presence of energy metabolic defects with the functional alterations associated with brain aging and with the pathogenesis of neurodegenerative disorders such as Alzheimers disease. Within this context we tested the ability of insulin to regulate the amyloid precursor protein (APP) processing in SH‐SY5Y neuroblastoma cells. Our findings show that insulin promotes APP metabolism by a glucose‐independent mechanism. We demonstrate a novel intracellular pathway that increases the rate of secretion of soluble APP through the activity of phosphati‐dyl‐inositol 3 kinase (PI3‐K). This pathway, downstream of insulin receptor tyrosine kinase activity, does not involve either the activation of protein kinase C or the mitogen‐activated protein kinase (MAP‐K) pathway. Because of the physiological role of PI3‐K in the translocation of glucose transporter‐containing vesicles, we speculate that PI3‐K involvement in APP metabolism may act at the level of vesicular trafficking.—Solano, D. C., Sironi, M., Bon‐fini, C., Solerte, S. B., Govoni, S., Racchi, M. Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase‐dependent pathway. FASEBJ. 14, 1015–1022 (2000)

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Abstract: Active uptake of 3,4‐dihydroxyphenylethylamine (dopamine) is sodium‐ and temperature‐dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+‐dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low‐ and high‐affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.

Cytosol protein kinase C downregulation in fibroblasts from Alzheimer's disease patients

We attempted to determine whether changes in protein kinase C (PKC) activity in Alzheimers disease (AD) brains are also present in cultured skin fibroblasts from living patients. Biopsies collected from shoulder skin were transferred to culture plates with an appropriate growth medium, and histone-directed PKC activity as well as phorbol ester binding were individually determined in soluble and particulate fractions prepared from AD and non-AD fibroblast cell lines. Binding experiments indicated that PKC was unevenly distributed between cytosol (78%) and particulate (22%). The Bmax values for phorbol ester binding in soluble and particulate fractions were similar in AD and non-AD patients. Kd values in the cytosol were 94% higher in AD patients, indicating lower affinity of the enzyme for the ligand. Accordingly, the soluble PKC activity was 30% lower in AD patients. The data suggest that the changes in PKC phosphorylating activity represent a diffuse cellular defect in AD and are not confined to the brain. The alterations of the enzyme may participate in the disregulation in processing of β-amyloid precursor protein in AD.

Afferent fibers mediate the increase of met-enkephalin elicited in rat spinal cord by localized pain

Abstract Met‐enkephalin levels were measured in various spinal cord regions of rats chronically suffering from the inflammation of a single paw following a treatment with Freunds adjuvant. The results indicate that chronic localized pain induces a selective increase of met‐enkephalin immunoreactive material (ME‐IR) in the dorsal horn of the spinal cord segment which receives a direct projection from the inflamed paw. In order to gain information on the functional meaning of these data, either the plexus brachialis or the sciatic nerve were sectioned peripherally before inducing inflammation. Denervation prevented the increase of ME‐IR concentration induced by the injection of Freunds adjuvant. Our observations suggest that chronic localized pain in a limb induces a change in ME‐IR content which is selective for the spinal cord segment receiving a direct projection from the inflamed paw. This increase depends on an intact innervation.

Members of the JAK/STAT proteins are expressed and regulated during development in the mammalian forebrain.

The presence and activation of members of the Janus Kinases/ Signal Transducers and Activator of Transcription proteins in response to specific cytokines is currently the focus of intense investigation in the hematopoietic system. Although some evidence suggests that cytokines might play an important role in brain development and brain pathologies, very limited information is available on the presence of the JAK/ STAT proteins in the Central Nervous System. Here we provide Western blot and immunohystochemistry data on the presence of Jak2 in vivo in the immature brain, its expression being greater in early stages of the embryonic life and gradually diminishing towards adulthood. Conversely, Jak1 was found expressed at a lower level compared to Jak2 and not modulated during brain maturation. Western blot data also show that specific members of the STAT family, the cytoplasmic substrates of the Janus Kinases, are present in vivo and that the extent of their expression is modulated differently at various stages. In particular, Stat6 protein levels were markedly attenuated at advanced stages of differentiation, as well as in the adult brain, with respect to early embryonic life. On the contrary, Stat3 levels did not vary. Analysis of Stat1 and Stat5 proteins showed a more complex expression pattern. These data indicate that members of the JAK/ STAT proteins are present and modulated in vivo in the embryonic and postnatal brain, therefore supporting their role in the modulation of gene expression during the different stages of brain maturation. J. Neurosci. Res. 54:320–330, 1998.

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

Impairment of brain neurotransmitter receptors in aged rats.

Dopamine and gamma-aminobutyric acid (GABA) receptor functions have been measured in various brain areas of aged rats. [3H] Spiroperidol binding is decreased in various dopaminergic brain areas, particularly in striatum and tuberculum olfactorium. In striatum the number of binding sites for [3H] spiroperidol is similar in both groups of animals, while the affinity is reduced in senescent rats. Moreover, in the pituitary a 50% increase of [3H] spiroperidol binding was detected in the group of senescent animals. On the other hand, [3H] GABA binding is significantly decreased in substantia nigra and hypothalamus of aged rats, while it is unmodified in cerebral cortex, cerebellum, striatum and nucleus accumbens.

Protein kinase C activity, translocation, and conventional isoforms in aging rat brain.

Protein kinase C was studied in various brain areas in aging Wistar rats. Histone-directed kinase activity from the cortex, hippocampus and cerebellum did not change with aging. Using purified protein B-50 as a substrate, between 3 and 8 months a decrease in in vitro phosphorylation was detected in the membrane fraction of the cortex but after this age values remained stable. In hippocampal membranes, B-50 phosphorylation was increased in aged rats. PKC translocation was impaired in aged rats in both the cortex and the hippocampus. PKC alpha and beta mRNA decreased in the cortex between 3 and 8 months with no further decline in aged animals. Hippocampal mRNA for calcium-dependent PKC isoforms was not modified during aging, as assessed by Northern and in situ hybridization. Western blot analysis revealed a change in PKC gamma protein only, which was increased in hippocampal membranes from aged rats. The data indicate that the key PKC function that is impaired in aged rats is enzyme translocation irrespective of the brain area investigated.

Age-related alteration of PKC, a key enzyme in memory processes: physiological and pathological examples.

Brain aging is characterized by a progressive decline of the cognitive and memory functions. It is becoming increasingly clear that protein phosphorylation and, in particular, the activity of the calcium-phospholipid-dependent protein kinase C (PKC) may be one of the fundamental cellular changes associated with memory function. PKC is a multigene family of enzymes highly expressed in brain tissues. The activation of kinase C is coupled with its translocation from the cytosol to different intracellular sites and recent studies have demonstrated the key role played by several anchoring proteins in this mechanism. PKC-phosphorylating activity appears to be impaired during senescence at brain level in a strain-dependent fashion in rodents. Whereas the levels of the various isoforms do not show age-related alterations, the enzyme translocation upon phorbol-ester treatment is deficitary among all strains investigated. Anchoring proteins may contribute to this activation deficit. We discuss also modifications of the PKC system in Alzheimers disease that may be related to pathological alterations in neurotransmission. A better insight of the different factors controlling brain-PKC activation may be important not only for elucidating the molecular basis of neuronal transmission, but also for identifying new approaches for correcting or even preventing age-dependent changes in brain function.