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Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor (PPAR)-γ agonists commonly used as insulin-sensitizing drugs for the treatment of type 2 diabetes. In the last decade, PPAR-γ agonists have received increasing attention for their neuroprotective properties displayed in a variety of neurodegenerative diseases, including Parkinson's disease (PD), likely related to the anti-infammatory activity of these compounds. Recent studies indicate that neuroinflammation, specifically reactive microglia, plays important roles in PD pathogenesis. Moreover, after the discovery of infiltrating activated Limphocytes in the substantia nigra (SN) of PD patients, most recent research supports a role of immune-mediated mechanisms in the pathological process leading to chronic neuroinflammation and dopaminergic degeneration. PPAR-γ are highly expressed in cells of both central and peripheral immune systems, playing a pivotal role in microglial activation as well as in monocytes and T cells differentiation, in which they act as key regulators of immune responses. Here, we review preclinical evidences of PPAR-γ-induced neuroprotection in experimental PD models and highlight relative anti-inflammatory mechanisms involving either central or peripheral immunomodulatory activity. Specific targeting of immune functions contributing to neuroinflammation either directly (central) or indirectly (peripheral) may represent a novel therapeutic approach for disease modifying therapies in PD.

The motor symptoms of Parkinson's disease (PD) are primarily due to the degeneration of the dopaminergic neurons in the nigrostriatal pathway. However, several other brain areas and neurotransmitters other than dopamine such as noradrenaline, 5-hydroxytryptamine and acetylcholine are affected in the disease. Moreover, adenosine because of the extensive interaction of its receptors with the dopaminergic system has been implicated in the pathophysiology of the disease. Based on the involvement of these non-dopaminergic neurotransmitters in PD and the sometimes severe adverse effects that limit the mainstay use of dopamine-based anti-parkinsonian treatments, recent assessments have called for a broadening of therapeutic options beyond the traditional dopaminergic drug arsenal. In this review we describe the interactions between dopamine and adenosine receptors that underpin the pre-clinical and clinical rationale for pursuing adenosine A(2A) receptor antagonists as symptomatic and potentially neuroprotective treatment of PD. The review will pay particular attention to recent results regarding specific A(2A) receptor-receptor interactions and recent findings identifying urate, the end product of purine metabolism, as a novel prognostic biomarker and candidate neuroprotectant in PD.

There are still many controversies about the role of leptin in reproductive function and sexual development. We recently demonstrated that leptin receptors are expressed in rodent Leydig cells and that leptin has inhibitory effects on hCG-stimulated testosterone production by adult rat Leydig cells in culture. In this study, we evaluated the expression of leptin receptor (Ob-R) in rat testes from gestational to adult age in comparison with the pattern of expression of relaxin-like factor (RLF), a specific marker of Leydig cell differentiation status. Immunohistochemical analysis showed that, in prenatal life, Ob-R immunoreactivity was absent at early embryonic ages (E14.5) and appeared at a late embryonic age (E19.5); in postnatal life, immunoreactivity was evident only after sexual maturation (35-, 60-, and 90-days old), whereas it was absent in testes from sexually immature rats (7-, 14-, and 21-days old). Immunoreaction was always confined to Leydig cells and no signal of Ob-R was detected within the tubules. The pattern of expression of Ob-R during testicular development was similar with that of RLF immunoreactivity, which was present in mature fetal as well as adult-type Leydig cells. In contrast with the findings in the testis, in the hypothalamus, the immunohistochemical pattern of Ob-R was very similar between pre- and postpubertal life. Reverse transcription-polymerase chain reaction studies showed that Ob-R expression was present in embryonic, prepubertal, and adult rat testes; semiquantitative analysis showed that mRNA levels were much higher in late versus early embryonic testes, as well as in mature adults versus sexually immature testes, with a gradual increase from younger to older ages. Functional studies showed that, while leptin (150 ng/ml) significantly inhibited hCG-stimulated testosterone production in adult rat Leydig cells (46% reduction; P0.01), it did not modify prepubertal rat Leydig cells steroidogenic function in vitro. In conclusion, we showed that, in rat testis, Ob-R expression is characteristic of mature Leydig cells (fetal and adult type) and it is functional in adult but not prepubertal life.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450-17alpha messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men.

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.

Marketed drugs for Parkinson’s disease (PD) treat disease motor symptoms but are ineffective in stopping or slowing disease progression. In the quest of novel pharmacological approaches that may target disease progression, drug-repurposing provides a strategy to accelerate the preclinical and clinical testing of drugs already approved for other medical indications. Here, we targeted the inflammatory component of PD pathology, by testing for the first time the disease-modifying properties of the immunomodulatory imide drug (IMiD) pomalidomide in a translational rat model of PD neuropathology based on the intranigral bilateral infusion of toxic preformed oligomers of human α-synuclein (H-αSynOs). The neuroprotective effect of pomalidomide (20 mg/kg; i.p. three times/week 48 h apart) was tested in the first stage of disease progression by means of a chronic two-month administration, starting 1 month after H-αSynOs infusion, when an already ongoing neuroinflammation is observed. The intracerebral infusion of H-αSynOs induced an impairment in motor and coordination performance that was fully rescued by pomalidomide, as assessed via a battery of motor tests three months after infusion. Moreover, H-αSynOs-infused rats displayed a 40–45% cell loss within the bilateral substantia nigra, as measured by stereological counting of TH + and Nissl-stained neurons, that was largely abolished by pomalidomide. The inflammatory response to H-αSynOs infusion and the pomalidomide treatment was evaluated both in CNS affected areas and peripherally in the serum. A reactive microgliosis, measured as the volume occupied by the microglial marker Iba-1, was present in the substantia nigra three months after H-αSynOs infusion as well as after H-αSynOs plus pomalidomide treatment. However, microglia differed for their phenotype among experimental groups. After H-αSynOs infusion, microglia displayed a proinflammatory profile, producing a large amount of the proinflammatory cytokine TNF-α. In contrast, pomalidomide inhibited the TNF-α overproduction and elevated the anti-inflammatory cytokine IL-10. Moreover, the H-αSynOs infusion induced a systemic inflammation with overproduction of serum proinflammatory cytokines and chemokines, that was largely mitigated by pomalidomide. Results provide evidence of the disease modifying potential of pomalidomide in a neuropathological rodent model of PD and support the repurposing of this drug for clinical testing in PD patients.

The accumulation of aggregated &alpha, synuclein (&alpha, Syn) is a hallmark of Parkinson&rsquo, s disease (PD). Current evidence indicates that small soluble &alpha, Syn oligomers (&alpha, SynOs) are the most toxic species among the forms of &alpha, Syn aggregates, and that size and topological structural properties are crucial factors for &alpha, SynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human &alpha, SynO (H-&alpha, SynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-&alpha, SynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-&alpha, SynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated &alpha, Syn (p129-&alpha, Syn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to &alpha, synOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-&alpha, SynOs triggered p129-&alpha, Syn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-&alpha, SynOs inhibited the phagocytic function of microglia. H-&alpha, synOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-&alpha, SynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies.

Parkinson’s disease (PD) is a complex pathology causing a plethora of non-motor symptoms besides classical motor impairments, including cognitive disturbances. Recent studies in the PD human brain have reported microgliosis in limbic and neocortical structures, suggesting a role for neuroinflammation in the development of cognitive decline. Yet, the mechanism underlying the cognitive pathology is under investigated, mainly for the lack of a valid preclinical neuropathological model reproducing the disease’s motor and non-motor aspects. Here, we show that the bilateral intracerebral infusion of pre-formed human alpha synuclein oligomers (H-αSynOs) within the substantia nigra pars compacta (SNpc) offers a valid model for studying the cognitive symptoms of PD, which adds to the classical motor aspects previously described in the same model. Indeed, H-αSynOs-infused rats displayed memory deficits in the two-trial recognition task in a Y maze and the novel object recognition (NOR) test performed three months after the oligomer infusion. In the anterior cingulate cortex (ACC) of H-αSynOs-infused rats the in vivo electrophysiological activity was altered and the expression of the neuron-specific immediate early gene (IEG) Npas4 (Neuronal PAS domain protein 4) and the AMPA receptor subunit GluR1 were decreased. The histological analysis of the brain of cognitively impaired rats showed a neuroinflammatory response in cognition-related regions such as the ACC and discrete subareas of the hippocampus, in the absence of any evident neuronal loss, supporting a role of neuroinflammation in cognitive decline. We found an increased GFAP reactivity and the acquisition of a proinflammatory phenotype by microglia, as indicated by the increased levels of microglial Tumor Necrosis Factor alpha (TNF-α) as compared to vehicle-infused rats. Moreover, diffused deposits of phospho-alpha synuclein (p-αSyn) and Lewy neurite-like aggregates were found in the SNpc and striatum, suggesting the spreading of toxic protein within anatomically interconnected areas. Altogether, we present a neuropathological rat model of PD that is relevant for the study of cognitive dysfunction featuring the disease. The intranigral infusion of toxic oligomeric species of alpha-synuclein (α-Syn) induced spreading and neuroinflammation in distant cognition-relevant regions, which may drive the altered neuronal activity underlying cognitive deficits.

Aggregates of alpha-synuclein (αSyn) have been described in Parkinson's disease (PD) patients, and recent evidence has suggested that the most toxic αSyn species in PD are small soluble aggregates including oligomers, prefibrils, protofibrils. The physiological function of αSyn is still highly debated, with a possible role in synaptic vesicle trafficking and release at the presynaptic compartment, and in the regulation of gene expression in the nucleus. Emerging evidence indicate that most of αSyn functions are related with the crucial ability to bind biological membranes, which is associated with structural conversion from a disordered monomer to an α-helical enriched structure. Conformational properties of αSyn can be modulated by a number of factors including post-translational modifications, gene duplication and triplication-driven overexpression, single point mutations, environmental changes, which affect membrane binding and the protein propensity to aggregate in toxic species. The recognized toxic role of αSyn in PD has laid the rational for purposing of αSyn-based, neuropathologically relevant preclinical models of PD. Different approaches have led to the establishment of transgenic models, viral vector-based models, and more recently models based on the intracerebral inoculation of exogenous αSyn preformed fibrils/oligomers. Here, we overview and compare viral vector-based models of αSyn overexpression and models obtained by direct intracerebral infusion of in vitro preformed αSyn species. The advantages and pitfalls associated with these different approaches are discussed.

Parkinson&rsquo, s disease (PD) is considered a synucleinopathy because of the intraneuronal accumulation of aggregated &alpha, synuclein (&alpha, Syn). Recent evidence points to soluble &alpha, Syn-oligomers (&alpha, SynO) as the main cytotoxic species responsible for cell death. Given the pivotal role of &alpha, Syn in PD, &alpha, Syn-based models are crucial for the investigation of toxic mechanisms and the identification of new therapeutic targets in PD. By using a metabolomics approach, we evaluated the metabolic profile of brain and serum samples of rats infused unilaterally with preformed human &alpha, SynOs (H&alpha, SynOs), or vehicle, into the substantia nigra pars compacta (SNpc). Three months postinfusion, the striatum was dissected for striatal dopamine (DA) measurements via High Pressure Liquid Chromatography (HPLC) analysis and mesencephalon and serum samples were collected for the evaluation of metabolite content via gas chromatography mass spectrometry analysis. Multivariate, univariate and correlation statistics were applied. A 40% decrease of DA content was measured in the H&alpha, SynO-infused striatum as compared to the contralateral and the vehicle-infused striata. Decreased levels of dehydroascorbic acid, myo-inositol, and glycine, and increased levels of threonine, were found in the mesencephalon, while increased contents of fructose and mannose, and a decrease in glycine and urea, were found in the serum of H&alpha, SynO-infused rats. The significant correlation between DA and metabolite content indicated that metabolic variations reflected the nigrostriatal degeneration. Collectively, the metabolomic fingerprint of H&alpha, SynO-infused rats points to an increase of oxidative stress markers, in line with PD neuropathology, and provides hints for potential biomarkers of PD.