Federica Bono

Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinsons disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our “in vitro” studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, “in vivo” studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the “in situ” visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT.

Nicotine-Induced Structural Plasticity in Mesencephalic Dopaminergic Neurons Is Mediated by Dopamine D3 Receptors and Akt-mTORC1 Signaling

Although long-term exposure to nicotine is highly addictive, one beneficial consequence of chronic tobacco use is a reduced risk for Parkinson’s disease. Of interest, these effects both reflect structural and functional plasticity of brain circuits controlling reward and motor behavior and, specifically, recruitment of nicotinic acetylcholine receptors (nAChR) in mesencephalic dopaminergic neurons. Because the underlying cellular mechanisms are poorly understood, we addressed this issue with use of primary cultures of mouse mesencephalic dopaminergic neurons. Exposure to nicotine (1–10 μM) for 72 hours in vitro increased dendritic arborization and soma size in primary cultures. These effects were blocked by mecamylamine and dihydro-β-erythroidine, but not methyllycaconitine. The involvement of α4β2 nAChR was supported by the lack of nicotine-induced structural remodeling in neurons from α4 null mutant mice (KO). Challenge with nicotine triggered phosphorylation of the extracellular signal-regulated kinase (ERK) and the thymoma viral proto-oncogene (Akt), followed by activation of the mammalian target of rapamycin complex 1 (mTORC1)-dependent p70 ribosomal S6 protein kinase. Upstream pathway blockade using the phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] resulted in suppression of nicotine-induced phosphorylations and structural plasticity. These effects were dependent on functional DA D3 receptor (D3R), because nicotine was inactive both in cultures from D3R KO mice and after pharmacologic blockade with D3R antagonist trans-N-4-2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethylcyclohexyl-4-quinolinecarboxamide (SB-277011-A) (50 nM). Finally, exposure to nicotine in utero (5 mg/kg/day for 5 days) resulted in increased soma area of DAergic neurons of newborn mice, effects not observed in D3 receptor null mutant mice mice. These findings indicate that nicotine-induced structural plasticity at mesencephalic dopaminergic neurons involves α4β2 nAChRs together with dopamine D3R-mediated recruitment of ERK/Akt-mTORC1 signaling.

Pre-synaptic dopamine D(3) receptor mediates cocaine-induced structural plasticity in mesencephalic dopaminergic neurons via ERK and Akt pathways.

J. Neurochem. (2012) 120, 765–778.

The D3 dopamine receptor: From structural interactions to function.

Novel structural and functional aspects of the dopamine (DA) D3 receptors (D3R) have been recently described. D3R expressed in dopaminergic neurons have been classically considered to play the role of autoreceptors inhibiting, as the D2R, DA release. However, evidence for D3R-mediated neurotrophic and neuroprotective effects on DA neurons suggests their involvement in preventing pathological alterations leading to neurodegeneration. On the other hand, given its localization and functional role at postsynaptic striatal levels, the D3R may also be involved in the pathogenesis of movement disorders and psychiatric diseases. Functional interactions of D3R with other receptor systems are crucial for the modulation of several physiological events. On this line, the discovery that the D3R can form heteromers with other receptors has opened the possibility of uncover novel molecular mechanisms of brain functions and dysfunctions. This paper summarizes the functional and physical interactions of D3R with other receptors both at pre-synaptic sites, where it is co-expressed with the D2R and nicotinic receptors, and at post-synaptic sites where it interacts with the DA D1 receptors (D1R). The biochemical and functional properties of the D1R-D3R heteromer will be especially discussed. Both D1R and D3R have been in fact implicated in several disorders, including schizophrenia and motor dysfunctions. Therefore, the D1R-D3R heteromer may represent a potential drug target for the treatment of these diseases.

Shp-2 knockdown prevents l-dopa-induced dyskinesia in a rat model of Parkinson's disease

Dyskinesia, the major side effect of l‐dopa therapy in PD, is mainly associated with nonphysiological stimulation of denervated receptors in the striatum. In particular, DA D1 receptor‐mediated aberrant extracellular signal‐regulated protein kinases 1 and 2 activation have been associated with striatal changes leading to dyskinesia. We recently identified the tyrosine phosphatase Shp‐2 as a crucial effector transmitting D1 receptor signaling to extracellular signal‐regulated protein kinases 1 and 2 activation and reported the involvement of the D1 receptor/Shp‐2/extracellular signal‐regulated protein kinases 1 and 2 pathway in the development of l‐dopa‐induced dyskinesia.

Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma.

Caveolins (Cav-1, -2 and -3) and Cavins (Cavin-1, -2, -3 and -4) are two protein families controlling the biogenesis and function of caveolae, plasma membrane omega-like invaginations representing the primary site of important cellular processes like endocytosis, cholesterol homeostasis and signal transduction. Caveolae are especially abundant in fat tissue, playing a consistent role in a number of processes, such as the insulin-dependent glucose uptake and transmembrane transport of lipids underlying differentiation, maintenance and adaptive hypertrophy of adipocytes. Based on this premise, in this work we have investigated the expression of caveolar protein components in liposarcoma (LPS), an adipocytic soft tissue sarcoma affecting adults categorized in well-differentiated, dedifferentiated, myxoid and pleomorphic histotypes. By performing an extensive microarray data analysis followed by immunohistochemistry on human LPS tumors, we demonstrated that Cav-1, Cav-2 and Cavin-1 always cluster in all the histotypes, reaching the highest expression in well-differentiated LPS, the least aggressive of the malignant forms composed by tumor cells with a morphology resembling mature adipocytes. In vitro experiments carried out using two human LPS cell lines showed that the expression levels of Cav-1, Cav-2 and Cavin-1 proteins were faintly detectable during cell growth, becoming consistently increased during the accumulation of intracellular lipid droplets characterizing the adipogenic differentiation. Moreover, in differentiated LPS cells the three proteins were also found to co-localize and form molecular aggregates at the plasma membrane, as shown via immunofluorescence and immunoprecipitation analysis. Overall, these data indicate that Cav-1, Cav-2 and Cavin-1 may be considered as reliable markers for identification of LPS tumors characterized by consistent adipogenic differentiation.

Mitochondria and α-Synuclein: Friends or Foes in the Pathogenesis of Parkinson’s Disease?

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.

Dopamine D3 and acetylcholine nicotinic receptor heteromerization in midbrain dopamine neurons: Relevance for neuroplasticity

Activation of nicotinic acetylcholine receptors (nAChR) promotes the morphological remodeling of cultured dopamine (DA) neurons, an effect requiring functional DA D3 receptors (D3R). The aim of this study was to investigate the mechanisms mediating D3R-nAChR cross-talk in the modulation of DA neuron structural plasticity. By using bioluminescence resonance energy transfer2 (BRET2) and proximity ligation assay (PLA), evidence for the existence of D3R-nAChR heteromers has been obtained. In particular, BRET2 showed that the D3R directly and specifically interacts with the β2 subunit of the nAChR. The D3R-nAChR complex was also identified in cultured DA neurons and in mouse Substantia Nigra/Ventral Tegmental Area by PLA. Cell permeable interfering peptides, containing highly charged amino acid sequences from the third intracellular loop of D3R (TAT-D3R) or the second intracellular loop of the β2 subunit (TAT-β2), were developed. Both peptides, but not their scrambled counterparts, significantly reduced the BRET2 signal generated by D3R-GFP2 and β2-Rluc. Similarly, the PLA signal was undetectable in DA neurons exposed to the interfering peptides. Moreover, interfering peptides abolished the neurotrophic effects of nicotine on DA neurons. Taken together these data first demonstrate that a D3R-nAChR heteromer is present in DA neurons and represents the functional unit mediating the neurotrophic effects of nicotine.

Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling

The antiparkinsonian ropinirole and pramipexole are D3 receptor- (D3R-) preferring dopaminergic (DA) agonists used as adjunctive therapeutics for the treatment resistant depression (TRD). While the exact antidepressant mechanism of action remains uncertain, a role for D3R in the restoration of impaired neuroplasticity occurring in TRD has been proposed. Since D3R agonists are highly expressed on DA neurons in humans, we studied the effect of ropinirole and pramipexole on structural plasticity using a translational model of human-inducible pluripotent stem cells (hiPSCs). Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084 and by the mTOR pathway kinase inhibitors LY294002 and rapamycin. All treatments were also effective in attenuating the D3R-dependent increase of p70S6-kinase phosphorylation. Immunoneutralisation of BDNF, inhibition of TrkB receptors, and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity, suggesting a critical interaction between BDNF and D3R signaling pathways. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpin their reliability for characterization of pharmacological agents acting via dopaminergic mechanisms.

Exploring pre-degenerative alterations in humans using induced pluripotent stem cell-derived dopaminergic neurons

Understanding the cellular and molecular mechanisms underlying human neurological disorders is hindered by both the complexity of the disorders and the lack of suitable experimental models recapitulating key pathological features of the disease. This is a crucial issue since a limited understanding of pathogenic mechanisms precludes the development of drugs counteracting the progression of the disease. Among neurological disorders, Parkinson’s disease (PD) is likely caused by a variable combination of genetic and environmental factors leading to the progressive degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNp) (Kalia and Lang, 2015). Animal models and post-mortem studies have provided indications that multiple cellular processes may be altered in both familiar and sporadic PD pathogenesis including protein folding and aggregation, protein membrane trafficking and mitochondrial function (Kalia and Lang, 2015). However, the various genetic models of PD do not fully recapitulate the characteristics of human disease, while postmortem tissues from patients often represent the end stage of disease and may exhibit neurochemical alterations due to chronic pharmacological treatments. The possibility to reprogram human somatic cells into cells with pluripotent potential (human induced pluripotent stem cell, hiPSC) (Takahashi et al., 2007) and to differentiate hiPSC into any cell type, including neurons, thus provides an innovative approach to model neurological diseases. On this line, more information about the pathogenesis of PD has been recently provided by iPSC technology. In particular, important disease-related pathologic events such as aberrant autophagy and mitochondrial and cytoskeletal abnormalities have been described (Zhang et al., 2017), thus pointing to these models as a valuable tool for mimicking PD phenotype in vitro. More intriguingly, at present, hiPSC likely represents a unique experimental model for recognizing pre-degenerative neuronal dysfunctions that prelude the development of full-blown PD. The prerequisite for investigating pre-degenerative defects is a comprehensive elucidation of the physiological properties of hiPSC-derived DA neurons, especially focusing on the expression and function of “core” molecules playing a critical role in neuronal physiology. The iPSC technology overcomes the inability to directly examine living human neurons, thus providing a platform for investigating the properties of any proteins in their naturally occurring neuronal population. On this line, using hiPSC-derived neurons form healthy subjects, a pharmacological characterization of various ligand-and voltage-gate ion channels has been recently reported (Dage et al., 2014; Chatzidaki et al., 2015). Moreover, a physiological characterization of human midbrain DA neurons differentiated from hiPSC has been provided, showing the ability of neurons to synthetize and release DA and the capability to reuptake DA into the cells. In the same model, the electrophysiological properties of DA neurons and the neuronal mitochondrial homeostasis have been also examined (Hartfield et al., 2014).