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Parkinson's disease (PD), the most common neurodegenerative movement disorder, is characterized by the progressive loss of nigral dopamine neurons. The deposition of fibrillary aggregated α-synuclein in Lewy bodies (LB), that is considered to play a causative role in the disease, constitutes another key neuropathological hallmark of PD. We have recently described that synapsin III (Syn III), a synaptic phosphoprotein that regulates dopamine release in cooperation with α-synuclein, is present in the α-synuclein insoluble fibrils composing the LB of patients affected by PD. Moreover, we observed that silencing of Syn III gene could prevent α-synuclein fibrillary aggregation in vitro. This evidence suggests that Syn III might be crucially involved in α-synuclein pathological deposition. To test this hypothesis, we studied whether mice knock-out (ko) for Syn III might be protected from α-synuclein aggregation and nigrostriatal neuron degeneration resulting from the unilateral injection of adeno-associated viral vectors (AAV)-mediating human wild-type (wt) α-synuclein overexpression (AAV-hαsyn). We found that Syn III ko mice injected with AAV-hαsyn did not develop fibrillary insoluble α-synuclein aggregates, showed reduced amount of α-synuclein oligomers detected by in situ proximity ligation assay (PLA) and lower levels of Ser129-phosphorylated α-synuclein. Moreover, the nigrostriatal neurons of Syn III ko mice were protected from both synaptic damage and degeneration triggered by the AAV-hαsyn injection. Our observations indicate that Syn III constitutes a crucial mediator of α-synuclein aggregation and toxicity and identify Syn III as a novel therapeutic target for PD.

Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α-synuclein (α-syn), are neuropathological hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α-syn and modulates α-syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III. The in situ proximity ligation assay revealed the accumulation of numerous proteinase K-resistant neuropathological inclusions that contained both α-syn and syn III in tight association in the brain of affected subjects. Most strikingly, syn III was identified as a component of α-syn-positive fibrils in LB-enriched protein extracts from PD brains. Finally, a positive correlation between syn III and α-syn levels was detected in the caudate putamen of PD subjects. Collectively, these findings indicate that syn III is a crucial α-syn interactant and a key component of LB fibrils in the brain of patients affected by PD.

Histone deacetylation, together with altered acetylation of NF-κB/RelA, encompassing the K310 residue acetylation, occur during brain ischemia. By restoring the normal acetylation condition, we previously reported that sub-threshold doses of resveratrol and entinostat (MS-275), respectively, an activator of the AMP-activated kinase (AMPK)-sirtuin 1 pathway and an inhibitor of class I histone deacetylases (HDACs), synergistically elicited neuroprotection in a mouse model of ischemic stroke. To improve the translational power of this approach, we investigated the efficacy of MS-275 replacement with valproate, the antiepileptic drug also reported to be a class I HDAC blocker. In cortical neurons previously exposed to oxygen glucose deprivation (OGD), valproate elicited neuroprotection at 100 nmol/mL concentration when used alone and at 1 nmol/mL concentration when associated with resveratrol (3 nmol/mL). Resveratrol and valproate restored the acetylation of histone H3 (K9/18), and they reduced the RelA(K310) acetylation and the Bim level in neurons exposed to OGD. Chromatin immunoprecipitation analysis showed that the synergistic drug association impaired the RelA binding to the Bim promoter, as well as the promoter-specific H3 (K9/18) acetylation. In mice subjected to 60 min of middle cerebral artery occlusion (MCAO), the association of resveratrol 680 µg/kg and valproate 200 µg/kg significantly reduced the infarct volume as well as the neurological deficits. The present study suggests that valproate and resveratrol may represent a promising ready-to-use strategy to treat post-ischemic brain damage.

Parkinson’s disease (PD) is a movement disorder characterized by dopaminergic nigrostriatal neuron degeneration and the formation of Lewy bodies (LB), pathological inclusions containing fibrils that are mainly composed of α-synuclein. Dopaminergic neurons, for their intrinsic characteristics, have a high energy demand that relies on the efficiency of the mitochondria respiratory chain. Dysregulations of mitochondria, deriving from alterations of complex I protein or oxidative DNA damage, change the trafficking, size and morphology of these organelles. Of note, these mitochondrial bioenergetics defects have been related to PD. A series of experimental evidence supports that α-synuclein physiological action is relevant for mitochondrial homeostasis, while its pathological aggregation can negatively impinge on mitochondrial function. It thus appears that imbalances in the equilibrium between the reciprocal modulatory action of mitochondria and α-synuclein can contribute to PD onset by inducing neuronal impairment. This review will try to highlight the role of physiological and pathological α-synuclein in the modulation of mitochondrial functions.

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

Loss-of-function mutations in the progranulin (PGRN) gene are a common cause of familial frontotemporal lobar degeneration (FTLD). This age-related neurodegenerative disorder, characterized by brain atrophy in the frontal and temporal lobes and such typical symptoms as cognitive and memory impairment, profound behavioral abnormalities, and personality changes is thought to be related to connectome dysfunctions. Recently, PGRN reduction has been found to induce a behavioral phenotype reminiscent of FTLD symptoms in mice by affecting neuron spine density and morphology, suggesting that the protein can influence neuronal structural plasticity. Here, we evaluated whether a partial haploinsufficiency-like PGRN depletion, achieved by using RNA interference in primary mouse cortical neurons, could modulate GluN2B-containing N-methyl-d-aspartate (NMDA) receptors and tau phosphorylation, which are crucially involved in the regulation of the structural plasticity of these cells. In addition, we studied the effect of PGRN decrease on neuronal cell arborization both in the presence and absence of GluN2B-containing NMDA receptor stimulation. We found that PGRN decline diminished GluN2B-containing NMDA receptor levels and density as well as NMDA-dependent tau phosphorylation. These alterations were accompanied by a marked drop in neuronal arborization that was prevented by an acute GluN2B-containing NMDA receptor stimulation. Our findings support that PGRN decrease, resulting from pathogenic mutations, might compromise the trophism of cortical neurons by affecting GluN2B-contaning NMDA receptors. These mechanisms might be implicated in the pathogenesis of FTLD.

Non-metastatic glycoprotein melanoma protein B (GPNMB), also known as osteoactivin (OA) is expressed in a wide array of tumors and represents an emerging target for drug development. In this study, we investigated the role of GPNMB/OA in the progression of human metastatic DU145 and PC3 prostate cancer cells. GPNMB/OA contribution in PCa malignant phenotype has been analyzed by small interfering RNA-induced GPNMB/OA silencing. We found that following GPNMB/OA silencing the migration capability of both DU145 and PC3 cells, evaluated by using in vitro invasivity assay, as well as the metalloproteinases MMP-2 and MMP-9 activity were equally strongly inhibited. By contrast knocking down GPNMB/OA weakly attenuated cell proliferation rate of DU145, an effect that paralleled with an increase number of apoptotic cells. However, PC3 cell growth seems to be not affected by GPNMB/OA. Together, these data reveal that GPNMB/OA acts as a critical molecular mediator promoting the acquisition of the more aggressive, pro-metastatic phenotype distinctive of human DU145 and PC3 cell lines.

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

Synaptopathies are diseases with synapse defects as shared pathogenic features, encompassing neurodegenerative disorders such as Parkinson’s disease (PD). In sporadic PD, the most common age-related neurodegenerative movement disorder, nigrostriatal dopaminergic deficits are responsible for the onset of motor symptoms that have been related toα-synuclein deposition at synaptic sites. Indeed,α-synuclein accumulation can impair synaptic dopamine release and induces the death of nigrostriatal neurons. While in physiological conditions the protein can interact with and modulate synaptic vesicle proteins and membranes, numerous experimental evidences have confirmed that its pathological aggregation can compromise correct neuronal functioning. In addition, recent findings indicate thatα-synuclein pathology spreads into the brain and can affect the peripheral autonomic and somatic nervous system. Indeed, monomeric, oligomeric, and fibrillaryα-synuclein can move from cell to cell and can trigger the aggregation of the endogenous protein in recipient neurons. This novel “prion-like” behavior could further contribute to synaptic failure in PD and other synucleinopathies. This review describes the major findings supporting the occurrence ofα-synuclein pathology propagation in PD and discusses how this phenomenon could induce or contribute to synaptic injury and degeneration.

Synaptopathies are diseases with synapse defects as shared pathogenic features, encompassing neurodegenerative disorders such as Parkinson's disease (PD). In sporadic PD, the most common age-related neurodegenerative movement disorder, nigrostriatal dopaminergic deficits are responsible for the onset of motor symptoms that have been related to α-synuclein deposition at synaptic sites. Indeed, α-synuclein accumulation can impair synaptic dopamine release and induces the death of nigrostriatal neurons. While in physiological conditions the protein can interact with and modulate synaptic vesicle proteins and membranes, numerous experimental evidences have confirmed that its pathological aggregation can compromise correct neuronal functioning. In addition, recent findings indicate that α-synuclein pathology spreads into the brain and can affect the peripheral autonomic and somatic nervous system. Indeed, monomeric, oligomeric, and fibrillary α-synuclein can move from cell to cell and can trigger the aggregation of the endogenous protein in recipient neurons. This novel “prion-like” behavior could further contribute to synaptic failure in PD and other synucleinopathies. This review describes the major findings supporting the occurrence of α-synuclein pathology propagation in PD and discusses how this phenomenon could induce or contribute to synaptic injury and degeneration.

The role of dopamine D2 and D3 receptors (D2R/D3R), located on midbrain dopaminergic (DA) neurons, in the regulation of DA synthesis and release and in DA neuron homeostasis has been extensively investigated in rodent animal models. By contrast, the properties of D2R/D3R in human DA neurons have not been elucidated yet. On this line, the use of human-induced pluripotent stem cells (hiPSCs) for producing any types of cells has offered the innovative opportunity for investigating the human neuronal phenotypes at the molecular levels. In the present study, hiPSCs generated from human dermal fibroblasts were used to produce midbrain DA (mDA) neurons, expressing the proper set of genes and proteins typical of authentic, terminally differentiated DA neurons. In this model, the expression and the functional properties of the human D2R/D3R were investigated with a combination of biochemical and functional techniques. We observed that in hiPSC-derived mDA neurons, the activation of D2R/D3R promotes the proliferation of neuronal progenitor cells. In addition, we found that D2R/D3R activation inhibits nicotine-stimulated DA release and exerts neurotrophic effects on mDA neurons that likely occur via the activation of PI3K-dependent mechanisms. Furthermore, D2R/D3R stimulation counteracts both the aggregation of alpha-synuclein induced by glucose deprivation and the associated neuronal damage affecting both the soma and the dendrites of mDA neurons. Taken together, these data point to the D2R/D3R-related signaling events as a biochemical pathway crucial for supporting both neuronal development and survival and protection of human DA neurons.

Among neurobiological mechanisms underlying antidepressant properties of ketamine, structural remodeling of prefrontal and hippocampal neurons has been proposed as critical. The suggested mechanism involves downstream activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which trigger mammalian target of rapamycin (mTOR)-dependent structural plasticity via brain-derived neurotrophic factor (BDNF) and protein neo-synthesis. We evaluated whether ketamine elicits similar molecular events in dopaminergic (DA) neurons, known to be affected in mood disorders, using a novel, translational strategy that involved mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. Sixty minutes exposure to ketamine elicited concentration-dependent increases of dendritic arborization and soma size in both mouse and human cultures as measured 72 hours after application. These structural effects were blocked by mTOR complex/signaling inhibitors like rapamycin. Direct evidence of mTOR activation by ketamine was revealed by its induction of p70S6 kinase. All effects of ketamine were abolished by AMPA receptor antagonists and mimicked by the AMPA-positive allosteric modulator CX614. Inhibition of BDNF signaling prevented induction of structural plasticity by ketamine or CX614. Furthermore, the actions of ketamine required functionally intact dopamine D3 receptors (D3R), as its effects were abolished by selective D3R antagonists and absent in D3R knockout preparations. Finally, the ketamine metabolite (2R,6R)-hydroxynorketamine mimicked ketamine effects at sub-micromolar concentrations. These data indicate that ketamine elicits structural plasticity by recruitment of AMPAR, mTOR and BDNF signaling in both mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. These observations are of likely relevance to the influence of ketamine upon mood and its other functional actions in vivo.

Background The goal is to analyze the role of Osteactivin (OA) in human prostate adenocarcinoma (CaP) cell lines, DU145 and PC3. OA plays a role in proliferation, adhesion, differentiation and protein synthesis in normal and malignant cells. Further, the OA expression is specific of activated mature osteoblasts. Methods DU145 and PC3 were maintained under recommended conditions and treated, respectively, with 50 ng/mL for 8 days and with 100 ng/mL for 15 days of Nerve Growth Factor (NGF). The NGF-induced reduction of invasive capacity was assessed by the BioCoat Matrigel Invasion Chambers technique. The gene expression was evaluated by Q-RT-PCR, while the protein expression by indirect immunofluorescence and by western blot. Results NGF treatment of DU145 and PC3 induced a reduction of 95% and 78% of the invasive capacity. NGF treatment decreased OA expression at both mRNA and protein levels. The OA was secreted into the culture medium. Conclusions DU145 and PC3 cells expressed OA, that is strongly reduced by NGF treatment. Since it is known that OA is involved in the acquisition of the cell invasive capability, it could thus be hypothesized that OA could be a factor that contributes to the acquisition of invasive properties of PCa and could be proposed as a biomarker of cell tendency to metastatization.

Alpha-synuclein is a natively unfolded protein playing a key role in the regulation of several neuronal synaptic functions in physiological and pathological conditions. Many studies, over the past years, have shown that it is actively involved in PD pathophysiology. Alpha-synuclein is integrated in a complex network of neuronal processes through the interaction with cytosolic and synaptic proteins. Hence, it is not the sole α-synuclein pathology but its effects on diverse protein partners and specific cellular pathways in the membrane and/or cytosolic districts such as endoplasmic reticulum/Golgi, axonal and synaptic compartments of dopaminergic neurons, that may cause the onset of neuronal cell dysfunction and degeneration which are among the key pathological features of the PD brain. Here we summarize a series of experimental data supporting that α-synuclein aggregation may induce dysfunction and degeneration of synapses via these multiple mechanisms. Taken together, these data add new insights into the complex mechanisms underlying synaptic derangement in PD and other α-synucleinopathies. This article is part of a Special Issue entitled: Brain Integration.

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel(-/-)) mice developed a Parkinson's disease-like neuropathology with ageing. At 18 months of age, c-rel(-/-) mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel(-/-) mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel(-/-) mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel(-/-) mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel(-/-) mice may be a suitable model of Parkinson's disease.

Exposure to psychostimulants results in neuroadaptive changes of the mesencephalic dopaminergic system including morphological reorganization of dopaminergic neurons. Increased dendrite arborization and soma area were previously observed in primary cultures of mesencephalic dopaminergic neurons after 3-day exposure to dopamine agonists via activation of D(3) autoreceptors (D(3) R). In this work, we showed that cocaine significantly increased dendritic arborization and soma area of dopaminergic neurons from E12.5 mouse embryos by activating phosphorylation of extracellular signal-regulated kinase (ERK) and thymoma viral proto-oncogene (Akt). These effects were dependent on functional D(3) R expression because cocaine did not produce morphological changes or ERK/Akt phosphorylation neither in primary cultures of D(3) R mutant mice nor following pharmacologic blockade with D(3) R antagonists SB-277011-A and S-33084. Cocaine effects on morphology and ERK/Akt phosphorylation were inhibited by pre-incubation with the phosphatidylinositol 3-kinase inhibitor LY294002. These observations were corroborated in vivo by morphometrical assessment of mesencephalic dopaminergic neurons of P1 newborns exposed to cocaine from E12.5 to E16.5. Cocaine increased the soma area of wild-type but not of D(3) R mutant mice, supporting the translational value of primary culture. These findings indicate a direct involvement of D3R and ERK/Akt pathways as critical mediators of cocaine-induced structural plasticity, suggesting their involvement in psychostimulant addiction.

We report a novel mechanism for dopamine D(1) receptor (D(1) R)-mediated extracellular signal-regulated kinases (Erk) activation in rat striatum. Erk signaling depends on phosphorylation and dephosphorylation events mediated by specific kinases and phosphatases. The tyrosine phosphatase Shp-2, that is required for Erk activation by tyrosine kinase receptors, has been recently shown to regulate signaling downstream of few G protein-coupled receptors. We show that the D(1) R interacts with Shp-2, that D(1) R stimulation results in Shp-2 tyrosine phosphorylation and activation in primary striatal neuronal cultures and that D(1) R/Shp-2 interaction is required for transmitting D(1) R-dependent signaling to Erk1/2 activation. D(1) R-mediated Erk1/2 phosphorylation in cultured striatal neurons is in fact abolished by over-expression of the inactive Shp-2(C/S) mutant and by small interfering RNA-induced Shp-2 silencing. Moreover, by using selective inhibitors we show that both D(1) R-induced Shp-2 activation and Erk1/2 phosphorylation are dependent on the cyclic AMP/protein kinase A pathway and require Src. These results, which were substantiated also in transfected human embryonic kidney 293 cells, provide a novel mechanism by which to converge D(1) R signaling to the Erk pathway and suggest that Shp-2 or the D(1) R/Shp-2 interface could represent a potential drug target for disorders of dopamine transmission involving malfunctioning of D(1) R signaling.

This study was designed to test the hypothesis that improved mitochondrial biogenesis could help reducing ischemic cerebral injury. We found that levels of proliferator-activated receptor γ coactivator 1α and nuclear respiratory factor-1, mitochondrial DNA content and other markers of mitochondrial biogenesis and function were reduced in primary mouse cortical neurons under oxygen-glucose deprivation (OGD). The glycogen synthase kinase-3 (GSK-3) inhibitor SB216763 activated an efficient mitochondrial biogenesis program in control cortical neurons and counteracted the OGD-mediated mitochondrial biogenesis impairment. This was accompanied by the activation of an antioxidant response that reduced mitochondrial reactive oxygen species generation and ischemic neuronal damage. The in vitro effects of SB216763 were mimicked by two other structurally unrelated GSK-3 inhibitors. The protective effects of SB216763 on OGD-mediated neuronal damage were abolished in the presence of diverse mitochondrial inhibitors. Finally, when systemically administered in vivo, SB216763 reduced the infarct size and recovered the loss of mitochondrial DNA in mice subjected to permanent middle cerebral artery occlusion. We conclude that GSK-3 inhibition by SB216763 might pave the way of novel promising therapies aimed at stimulating the renewal of functional mitochondria and reducing reactive oxygen species-mediated damage in ischemic stroke.

J. Neurochem. (2011) 116, 588–605. Abstract Accumulation of misfolded proteins in the endoplasmic reticulum (ER) is the main event leading to the induction of the ER stress-related unfolded protein response (UPR). Recent postmortem evaluation, showing that the UPR pathway is activated in nigral dopaminergic neurons bearing α-synuclein inclusions in the brain of Parkinson’s disease (PD) patients, suggests that the activation of the UPR may be induced by the accumulation of α-synuclein. In this study, we show that the misfolded protein-sensor/UPR activator glucose-regulated protein 78/immunoglobulin heavy chain-binding protein was bound to α-synuclein and was increased in ‘in vitro’ and ‘in vivo’ models showing aggregated α-synuclein accumulation. Moreover, α-synuclein accumulation induced the expression of the UPR-related activating transcription factor 4/cAMP-responsive element-2. These findings indicate that activation of the UPR pathway in the PD brain is associated with α-synuclein accumulation occurring in part within the ER.

The widespread non-neuronal synthesis of acetylcholine (ACh) has changed the paradigm of ACh acting solely as a neurotransmitter. Indeed, the presence of ACh in many types of proliferating cells suggests a role for this neurotransmitter in the control of cell division. The parasympathetic system is a major pathway regulating micturition, but ACh-mediated control plays a more complex role than previously described, acting not only in the detrusor muscle, but also influencing detrusor function through the activity of urothelial muscarinic receptors. Here we investigated the role of muscarinic receptors in mediating cell proliferation in the human UROtsa cell line, which is a widely used experimental model to study urothelium physiology and pathophysiology. Our results demonstrate that UROtsa cells express the machinery for ACh synthesis and that muscarinic receptors, with the rank order of M3 > M2 > M5 > M1 = M4, are present and functionally linked to their known second messengers. Indeed, the cholinergic receptor agonist carbachol (CCh) (1–100 μM) concentration-dependently raised IP3 levels, reaching 66 ± 5% over basal. The forskolin-mediated adenylyl cyclase activation was reduced by CCh exposure (forskolin: 1.4 ± 0.14 pmol/ml; forskolin + 100 μM CCh: 0.84 ± 0.12 pmol/ml). CCh (1–100 μM) concentration-dependently increased UROtsa cell proliferation and this effect was inhibited by the non-selective antagonist atropine and the M3-selective antagonists darifenacin and J104129. Finally, CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation.

The relationship between β-amyloid (Aβ) and tau is not fully understood, though it is proposed that in the pathogenesis of Alzheimer's disease (AD) Aβ accumula- tion precedes and promotes tau hyperphosphorylation via activation of glycogen synthase kinase-3beta (GSK-3β). Both events contribute to learning and memory impair- ments. Modulation of γ-secretase activity has proved to reduce the Aβ burden and cognitive deficits in mouse models of AD, but its ability in reducing the tau pathology remains elusive. Chronic treatments with two γ-secretase modulators, ibuprofen and CHF5074, disclosed higher activity of CHF5074 in ameliorating brain plaque deposi- tion and spatial memory deficits in transgenic mice expressing human amyloid precursor protein (hAPP) with Swedish and London mutations (APPSL mice). The aim of our study was to investigate in APPSL mice the effect of the two compounds on the accumulation of native hyper- phosphorylated tau as well as on the GSK-3β signaling. CHF5074 was more effective than ibuprofen in reducing tau pathology, though both compounds decreased the GSK- 3β level and increased the GSK-3β inhibitory phosphory- lation near to the non-Tg values. The inhibition of GSK-3β appeared to be secondary to the reduction of Aβ generation as, differently from LiCl, CHF5074 reproduced its effect in hAPP-overexpressing neuroglioma cells, but not in wild- type primary neurons. Our data show that the novel γ- secretase modulator CHF5074 can fully reverse β-amyloid- associated tau pathology, thus representing a promising therapeutic agent for AD.

The authors tested the effects of 2-Br-ergocriptine (bromocriptine, CB-154), a drug which exerts a mixed agonist-antagonist activity on the dopaminergic receptors, in 12 patients with Huntington Chorea in a double-blind crossover trial. This treatment significantly reduced the abnormal involuntary movements and the disease severity in most of the patients. Subjects who were slightly disabled showed a better response than the ones with more severe degrees of disability.

Diverse nuclear factor-kappaB subunits mediate opposite effects of extracellular signals on neuron survival. While RelA is activated by neurotoxic agents, c-Rel drives neuroprotective effects. In brain ischaemia RelA and p50 factors rapidly activate, but how they associate with c-Rel to form active dimers and contribute to the changes in diverse dimer activation for neuron susceptibility is unknown. We show that in both cortical neurons exposed to oxygen glucose deprivation (OGD) and mice subjected to brain ischaemia, activation of p50/RelA was associated with inhibition of c-Rel/RelA dimer and no change p50/c-Rel. Targeting c-Rel and RelA expression revealed that c-Rel dimers reduced while p50/RelA enhanced neuronal susceptibility to anoxia. Activation of p50/RelA complex is known to induce the pro-apoptotic Bim and Noxa genes. We now show that c-Rel-containing dimers, p50/c-Rel and RelA/c-Rel, but not p50/RelA, promoted Bcl-xL transcription. Accordingly, the OGD exposure induced Bim, but reduced Bcl-xL promoter activity and decreased the content of endogenous Bcl-xL protein. These findings demonstrate that within the same neuronal cell, the balance between activation of p50/RelA and c-Rel-containing complexes fine tunes the threshold of neuron vulnerability to the ischaemic insult. Selective targeting of different dimers will unravel new approaches to limit ischaemia-associated apoptosis.

Nuclear factor-kappaB (NF-κB) has been proposed to serve a dual function as a regulator of neuron survival in pathological conditions associated with neurodegeneration. NF-κB is a transcription family of factors comprising five different proteins, namely p50, RelA/p65, c-Rel, RelB and p52, which can combine differently to form active dimers in response to external stimuli. Recent research shows that diverse NF-κB dimers lead to cell death or cell survival in neurons exposed to ischemic injury. While the p50/p65 dimer participates in the pathogenesis of post-ischemic injury by inducing pro-apoptotic gene expression, c-Rel-containing dimers increase neuron resistance to ischemia by inducing anti-apoptotic gene transcription. We present, in this report, the latest findings and consider the therapeutic potential of targeting different NF-κB dimers to limit ischemia-associated neurodegeneration.

s / Parkinsonism and Related Disorders 22 (2016) e149ee192 e189 Conclusions: The results indicate that the a-mannosidase activity present in the CSF is of lysosomal type and of brain origin. Furthermore, the intermediate form of a-mannosidase from plasma does not cross the bloodbrain-barrier. Thus CSF a-mannosidase activity might mirror the brain pathological changes linked to neurodegenerative disorders such as PD. P 6.058. SYNAPSIN III ALTERATIONS IN PARKINSON'S DISEASE Arianna Bellucci , Michela Zaltieri , Francesca Longhena , Gaia Faustini , Jessica Grigoletto , Gaia Favero , Stefania Castrezzati , Rita Rezzani , Marina Pizzi , Fabio Benfenati , Maria Grazia Spillantini , Cristina Missale , PierFranco Spano . Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy; Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genoa, Italy; Department of Clinical Neuroscience, University of Cambridge, Cambridge, United Kingdom; 5 IRCCS San Camillo Hospital for Neurorehabilitation (NHS-Italy), Venice, Italy Objectives: The main neuropathological hallmarks of Parkinson's disease (PD) are loss of nigro-striatal dopamine neurons and intraneuronal Lewy Bodies (LB), proteinaceous inclusions mainly composed by a-synuclein. Recently, we found that a-synuclein interacts and cooperates with a specific member of the synapsin phosphoprotein family: synapsin III, in the regulation of dopaminergic neuron function. The aim of this study was to investigate whether synapsin III alterations may be related to a-synuclein pathology in PD. We thus investigated the occurrence of alterations of synapsin III expression and distribution in “the brain of patients affected by PD and DLB (Human brain samples were kindly provided by the UK Parkinson's disease Brain Bank). Methods: We used molecular biology and immunohistochemical techniques as well as the “in situ” proximity ligation assay (PLA) to investigate the expression levels and the distribution of synapsin III in the PD brain. Results: By immunohistochemistry, we showed a marked accumulation of synapsin III in the caudate/putamen of patients affected by PD when compared to age matched controls. In addition, many LB-like structures in the substantia nigra of PD patients were found to be immunoreactive for synapsin III, that by confocal fluorescence microscopy resulted to be accumulated in the core of LB. By co-immunoprecipitation and “in situ” PLA we found that synapsin III directly interacted with a-synuclein and that these two proteins were co-redistributed in mice and human brains. Finally, western blot analysis showed significant alterations of synapsin III levels that correlated with alteration of a-synuclein in the caudate/putamen and substantia nigra of PD patients. Conclusions: Altogether, our data support a critical involvement of synapsin III in PD pathophysiology P 6.059. UNDERSTANDING THE DIFFERENTIAL REGULATION OF GBA AND GBAP1 EXPRESSION Valeria Rimoldi , Giulia Rovaris , Letizia Straniero , Gianni Pezzoli , Stefano Goldwurm , Giulia Sold a , Rosanna Asselta , Stefano Duga . Humanitas University, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; Universit a degli Studi di Milano, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; 3 Parkinson Institute, Istituti Clinici di Perfezionamento, Milano, Italy Objectives: GBA downregulation was demonstrated to have a crucial role in Parkinson's Disease. We previously showed that there is a miRNA-mediated regulatory circuit linking GBA and its pseudogene (GBAP1) expression and that they are coexpressed inmost tissues with a GBA/GBAP1 ratio ranging from 2 to 200. Furthermore, we demonstrated that GBAP1 levels are downregulated by the NMD pathway. GBA and GBAP1 share a similar genomic structure characterized by a proximal (P1) and a distal (P2) promoter but there are no data on the differential regulation at the transcriptional level. Hence, wewant to study the P1 and P2 promoters of both genes. Methods: We cloned 1kb of the 4 promoters in a luciferase vector and produced serial 5'-deletion constructs of each promoter. These plasmids were transfected in different cell lines. Results: We demonstrated that: P2 promoters are stronger than P1s, for both GBA and GBAP1; in HeLa cells, as expected, GBAP1 promoters are weaker than GBA ones, while in SH-SY5Y cells, the pseudogene P2 promoter has the highest activity; the TFEB binding sites seem to be the most important regulatory elements for P1 promoters. Conclusions: The transcriptional activity of both GBAP1 promoters, as assessed by luciferase assays, was unexpectedly higher compared to the mRNA levels detected in vivo. Besides differences in epigenetic regulation not faithfully modeled in our system, NMD is likely to be the major determinant of the relative expression of GBA/GBAP1 transcripts. P 6.061. STRUCTURAL ANALYSIS OF ALPHA-SYNUCLEIN OLIGOMERS VIA ANTIBODY FINGERPRINTING Lina Nilsson , Tohidul Islam , Irina Iakovleva , Kristoffer Br€annstr€ om, Anders Olofsson . Department of Chemistry, Umea, Sweden; Department of Medical Biochemistry and Biophysics, Umea

The combination of palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the family of the N-acylethanolamines, and the flavonoid luteolin has been found to exert neuroprotective activities in a variety of mouse models of neurological disorders, including brain ischemia. Indirect findings suggest that the two molecules can reduce the activation of mastocytes in brain ischemia, thus modulating crucial cells that trigger the inflammatory cascade. Though, no evidence exists about a direct effect of PEA and luteolin on mast cells in experimental models of brain ischemia, either used separately or in combination. In order to fill this gap, we developed a novel cell-based model of severe brain ischemia consisting of primary mouse cortical neurons and cloned mast cells derived from mouse fetal liver (MC/9 cells) subjected to oxygen and glucose deprivation (OGD). OGD exposure promoted both mast cell degranulation and the release of lactate dehydrogenase (LDH) in a time-dependent fashion. MC/9 cells exacerbated neuronal damage in neuron-mast cells co-cultures exposed to OGD. Likewise, the conditioned medium derived from OGD-exposed MC/9 cells induced significant neurotoxicity in control primary neurons. PEA and luteolin pre-treatment synergistically prevented the OGD-induced degranulation of mast cells and reduced the neurotoxic potential of MC/9 cells conditioned medium. Finally, the association of the two drugs promoted a direct synergistic neuroprotection even in pure cortical neurons exposed to OGD. In summary, our results indicate that mast cells release neurotoxic factors upon OGD-induced activation. The association PEA-luteolin actively reduces mast cell-mediated neurotoxicity as well as pure neurons susceptibility to OGD.

Preclinical data indicate a direct anti-tumor effect of zoledronic acid (ZA) outside the skeleton, but its molecular mechanism is still not completely clarified. The aim of this study was to investigate the anti-cancer effects of ZA in human breast cancer cell lines, suggesting that they may in part be mediated via the miR-21/PTEN/Akt signaling pathway. The effect of ZA on cell viability was measured by MTT assay, and cell death induction was analyzed using either a double AO/EtBr staining and M30 ELISA assay. A Proteome Profiler Human Apoptosis Array was executed to evaluate the molecular basis of ZA-induced apoptosis. Cell cycle analysis was executed by flow cytometry. The effect of ZA on miR-21 expression was quantified by qRT-PCR, and the amount of PTEN protein and its targets were analyzed by Western blot. ZA inhibited cell growth in a concentration- and time-dependent manner, through the activation of cell death pathways and arrest of cell cycle progression. ZA downregulated the expression of miR-21, resulting in dephosphorilation of Akt and Bad and in a significant increase of p21 and p27 proteins expression. These results were observed also in MDA-MB-231 cells, commonly used as an experimental model of bone metastasis of breast cancer. This study revealed, for the first time, an involvement of the miR-21/PTEN/Akt signaling pathway in the mechanism of ZA anti-cancer actions in breast cancer cells. We would like to underline that this pathway is present both in the hormone responsive BC cell line (MCF-7) as well as in a triple negative cell line (MDA-MB-231). Taken together these results reinforce the use of ZA in clinical practice, suggesting the role of miR-21 as a possible mediator of its therapeutic efficacy.

Parkinson's disease (PD) is a common neurodegenerative disorder with prominent loss of nigro-striatal dopaminergic neurons. The resultant dopamine (DA) deficiency underlies the onset of typical motor symptoms (MS). Nonetheless, individuals affected by PD usually show a plethora of nonmotor symptoms (NMS), part of which may precede the onset of motor signs. Besides DA neuron degeneration, a key neuropathological alteration in the PD brain is Lewy pathology. This is characterized by abnormal intraneuronal (Lewy bodies) and intraneuritic (Lewy neurites) deposits of fibrillary aggregates mainly composed of α-synuclein. Lewy pathology has been hypothesized to progress in a stereotypical pattern over the course of PD and α-synuclein mutations and multiplications have been found to cause monogenic forms of the disease, thus raising the question as to whether this protein is pathogenic in this disorder. Findings showing that the majority of α-synuclein aggregates in PD are located at presynapses and this underlies the onset of synaptic and axonal degeneration, coupled to the fact that functional connectivity changes correlate with disease progression, strengthen this idea. Indeed, by altering the proper action of key molecules involved in the control of neurotransmitter release and re-cycling as well as synaptic and structural plasticity, α-synuclein deposition may crucially impair axonal trafficking, resulting in a series of noxious events, whose pressure may inevitably degenerate into neuronal damage and death. Here, we provide a timely overview of the molecular features of synaptic loss in PD and disclose their possible translation into clinical symptoms through functional disconnection.

Recent findings indicate that skin-derived precursor cells (SKPs) of mouse dermis can differentiate in cells with neuronal-like morphology. However, direct evidence supporting the establishment of functional phenotype is missing. In the present study, SKP cells were obtained using published in vitro techniques and studied at 14- to 21-day differentiation, when neuronal-like morphology was observed. The experiment was repeated 39 times. Co-cultures with cortical astrocytes were also used to enhance the process of neural differentiation. Expression of GAP43 and light-chain MAP-2(c) but not markers of dendritic and synaptic terminal differentiation such as heavy-chain MAP-2(ab) and synapsin were observed. Voltage-clamp electrophysiology recordings showed potassium currents, but neither action potentials generation nor electrotonic response to exogenous administration of nicotine or kainic acid. These observations suggest that mouse SKPs do not differentiate into mature functional neurons, at least using the published methodologies for in vitro differentiation.

The nuclear transcription factors kappaB (NF-kappaB) function as key regulators of physiological processes in the central nervous system. Aberrant regulation of NF-kappaB can underlie neurological disorders associated with neurodegeneration. A large number of studies have reported a dual role of NF-kappaB in regulating neuron survival in pathological conditions. A recent progress in understanding the mechanisms responsible for opposite effects elicited by NF-kappaB in brain dysfunctions arises from the identification of diverse NF-kappaB complexes specifically involved in the mechanism of neuronal cell death or cell survival. We here discuss the latest findings and consider the therapeutic potential of targeting distinct NF-kappaB complexes for the treatment of neurodegenerative disorders and memory dysfunctions.

An increasing amount of evidence suggests that the family of nuclear factor κB (NF-κB) transcription factors plays an important role in synaptic plasticity and long-term memory formation. The present study investigated the regulation of NF-κB family members p50, p65/RelA, and c-Rel in the hippocampus in response to metabotropic glutamate receptor (mGluR) signaling. Activation of group I metabotropic glutamate receptors (GpI-mGluRs) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) resulted in a time-dependent increase in DNA binding activity of p50, p65, and c-Rel in area CA1 of the hippocampus. An antagonist of mGluR5, 2-Methyl-6-(phenylethynyl)pyridine, inhibited the DHPG-induced activation of NF-κB, whereas an antagonist of mGluR1, (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid, did not. Using a series of inhibitors, we investigated the signaling pathways necessary for DHPG-induced activation of NF-κB and found that they included the phosphatidyl inositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and p38-mitogen-activated protein kinase pathways. To determine the functional significance of mGluR-induced regulation of NF-κB, we measured long-term depression (LTD) of Schaffer-collateral synapses in the hippocampus of c-Rel knock-out mice. Early phase LTD was normal in c-rel−/−mice. However, late-phase LTD (>90 min) was impaired in c-rel−/−mice. The observations of this deficit in hippocampal synaptic plasticity prompted us to further investigate long-term memory formation in c-rel−/−mice. c-rel−/−mice exhibited impaired performance in a long-term passive avoidance task, providing additional evidence for c-Rel in long-term memory formation. These results demonstrate that the NF-κB transcription factor family is regulated by GpI-mGluRs in the hippocampus and that the c-Rel transcription factor is necessary for long-term maintenance of LTD and formation of long-term memory.

Beta-amyloid (Abeta) peptides are key proteins in the pathophysiology of Alzheimer's disease (AD). While Abeta42 aggregates very rapidly to form early diffuse plaques, supplemental Abeta40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor-kappaB (NF-kappaB) pathway in Abeta40-mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Abeta peptide induced a dose-dependent neuronal death, reduced the levels of the anti-apoptotic protein Bcl-XL, enhanced the cytosolic release of cytochrome c, and elicited the intracellular accumulation and secretion of Abeta42 oligomers. Moreover, Abeta40 activated the NF-kappaB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression. As inhibitors of the NF-kappaB pathway, we tested the capability of a double-stranded kappaB decoy oligonucleotide, the anti-inflammatory drug aspirin and the selective IkappaB kinase 2 inhibitor, AS602868, to modify the Abeta40-mediated effects. These treatments, transiently applied before Abeta exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The kappaB decoy also inhibited the Abeta-induced release of cytochrome c, restored the levels of Bcl-XL, and prevented intraneuronal accumulation and secretion of Abeta42. These results open up interesting perspectives on the development of novel strategies targeting out NF-kappaB p50/p65 dimers for pharmacological intervention in AD.

The aim of this work is to report a detailed study of the presence of α1 AR subtypes in human urinary bladder areas involved in the micturition (i.e. detrusor, trigone and neck), investigating whether or not there are differences between sexes. Methods Urinary bladder specimens were obtained as discarded tissue after cystectomy for vesical carcinoma (17 men and 14 women). Molecular characterization of α1 AR subtypes was done by semi-quantitative RT-PCR. The α1 AR protein expression was studied by saturation binding curves using the α1 AR antagonist 125I-HEAT and by western blot. Analysis of data was performed using the GraphPad PRISM 4 software. Results Results obtained indicated that α1 AR proteins were detectable in each bladder area. In both sexes, the detrusor and the neck expressed similar levels of α1 ARs: respectively, detrusor: 14.6 ± 1.2 in men and 13.1 ± 1.1 fmol/mg prot in women; neck: 16.9±3.2 in men and 17.5 ± 4.1 fmol/mg prot in women. In the trigone, significantly higher α1 ARs were found in women compared to men (20.6 ± 1.1 vs 11.7 ± 0.7 fmol/mg prot). Subtype analysis indicated that in women, each area was endowed with mRNA and protein encoding for each α1 AR subtype. The men detrusor expressed α1A and α1D ARs, while in the trigone and the neck, each subtype was present. Conclusions A selective distribution of α1 ARs did exist between sexes and between the different areas involved in micturition, supporting evidence which indicates a role of different α1 AR subtypes in the pathophysiology of the lower urinary tract symptoms.

Several studies proposed a role for α1 adrenoceptors (α1 ARs) in the ureteral physiology, indicating that they are present in the ureter; however, few studies have been made to identify α1 AR subtypes present in this area. Thus, this study was carried out to characterize the α1 AR subtype gene and protein expression in proximal, medial and distal region of the human ureter. Methods Molecular characterization of α1 AR subtypes was done by semi-quantitative RT-PCR. The α1 AR protein expression was studied by saturation binding curves using the α1 AR antagonist 125I-HEAT. Analysis of data was performed using the GraphPad PRISM 4 software. Results Analysis of saturation binding curves revealed an heterogeneous distribution of α1 AR binding sites; the Bmax for the distal ureter was indeed 52.5 ±5.4 fmol/mg prot, while a lower similar density of α1 ARs was demonstrated in the medial (25.2 ±1.7 fmol/mg prot) and proximal (23.4 ±0.4 fmol/mg prot) ureters. Molecular characterization of α1 AR subtypes indicated that each receptor was present, although with differences in term of the amount expressed. Conclusions Human ureter was endowed with each α1 AR subtype, although α1D and α1A ARs were prevalent over α1B ARs. Radioligand binding results revealed that there were no significant differences in the Kd between ureteral regions, while an heterogeneous distribution of α1 AR binding sites was detected, with the highest density of α1 ARs in the distal ureter and a lower similar density in the medial and proximal ureter.

Aim Several studies have proposed a role for α1 adrenoceptors (ARs) in ureteral physiology, indicating that they are present in the ureter; however, few studies have been done to identify α1 AR subtypes present in this area. Thus, this study was carried out to characterize the α1 AR subtype gene and protein expression in proximal, medial, and distal region of the human ureter. Methods Molecular characterization of α1 AR subtypes were analyzed by semi-quantitative RT-PCR. α1 AR protein expression was studied by saturation binding curves and by competition binding curves with selective antagonists. Analysis of data was performed using the GraphPad PRISM 4 software. Results Analysis of saturation binding curves revealed a heterogeneous distribution of α1 AR binding sites, the Bmax for the distal ureter was indeed 52.5 ± 5.4 fmol/mg prot, while a lower similar density of α1 ARs was demonstrated in the medial (25.2 ± 1.7 fmol/mg prot) and proximal (23.4 ± 0.4 fmol/mg prot) ureters. Molecular and pharmacological characterization of α1 AR subtypes indicated that each receptor was present, although with differences in terms of the amount expressed. Conclusions Human ureter was endowed with each α1 AR subtype, although α1D and α1A ARs were prevalent over α1B ARs. Radioligand binding results revealed that there were no significant differences in the Kd between ureteral regions, while a heterogeneous distribution of α1 AR binding sites was detected, with the highest density of α1 ARs in the distal ureter and a lower similar density in the medial and proximal ureters. Neurourol. Urodynam. 24:142–148, 2005. © 2005 Wiley-Liss, Inc.

Differentiated human NT2-N neurons were shown to express CCR5 and CXCR4 chemokine receptor mRNA and protein, and to be responsive to the chemokines CCL5 and CXCL12. Using cDNA microarray technology, CCL5 was found to induce a distinct transcriptional program, with reproducible induction of 46 and 9 genes after 2 and 8 hr of treatment, respectively. Conversely, downregulation of 20 and 7 genes was observed after 2 and 8 hr of treatment, respectively. Modulation of a selected panel of CCL5-responsive genes was also confirmed by quantitative RT-PCR and Western blot and compared to gene expression changes induced by CXCL12 treatment. Gene clustering identified distinct functional subsets of CCL5-responsive molecules, and a significant number of expressed sequence tags encoding unknown genes. CCL5-responsive genes comprise a significant number of enzymes, transcription factors, and miscellaneous molecules involved in neuronal survival and differentiation, including neurite outgrowth and synaptogenesis. Our results suggest that CCL5 biological functions might go beyond its recognized chemotactic activity in the central nervous system, in particular with regard to the control of neural plasticity events both during development and in postnatal life.

Nerve growth factor (NGF) is known to play a role as a circulating neurokine, integrating signals from the neuro-immuno-endocrine system. The ability of NGF to activate the pituitary-adrenocortical axis, together with the increase of its serum concentration in pregnancy and lactation, supports the hypothesis that NGF is secreted by the pituitary gland and plays a role as modulator of endocrine functions. Evidence obtained both in vitro and in vivo in experimental animal models suggests that lactotroph cells secrete both prolactin (PRL) and NGF. Furthermore, in previous studies we demonstrate that cell lines derived from dopamine (DA)-sensitive human prolactinomas express high levels of NGF messenger RNA and protein. On these basis, we studied serum NGF concentrations in female patients with microprolactinoma (n = 4) and in control women (n = 5). PRL and NGF were measured at the diagnosis, during the thyrotropin releasing hormone (TRH) test and after the therapy with DA D2 receptor agonist cabergoline (0.25 mg, twice a week). Results obtained suggested that hyperprolactinemia (70.3+/-8.4 ng/ml) paralleled markedly higher NGF levels (58.4+/-18.7 pg/ml) compared to controls (PRL 8.7+/-3.2 ng/ml, NGF 8.4+/-1.3 pg/ml). Serum concentrations of NGF and PRL during the TRH test were closely associated (r = 0.943, p0.01). Cabergoline therapy normalized PRL (7.9+/-3.6 ng/ml) and induced a significant decrease of NGF levels (12.5+/-4.9 pg/ml). In conclusions, data reported here indicated that, in human microprolactinomas, NGF is released in the bloodstream paralleling PRL-secretion and it is modulated by a neurotransmitter-regulated mechanism, since the normalization of PRL elicited by the DA D2 receptor agonist cabergoline induced a significant decrease of serum NGF as well.