Impaired dopamine homeostasis is an early event in the pathogenesis of Parkinson's disease. Generation of intracellular reactive oxygen species consequent to dopamine oxidation leads to mitochondrial dysfunction and eventually cell death. Alterations in the mitochondrial proteome due to dopamine exposure were investigated in the SH-SY5Y human neuroblastoma cell line. The combination of two orthogonal proteomic approaches, two-dimensional electrophoresis and shotgun proteomics (proteomeXchange dataset PXD000838), was used to highlight the specific pathways perturbed by the increase of intracellular dopamine, in comparison with those perturbed by a specific mitochondrial toxin (4-methylphenylpyridinium, MPP(+)), a neurotoxin causing Parkinsonism-like symptoms in animal models. Proteins altered by MPP(+) did not completely overlap with those affected by dopamine treatment. In particular, the MPP(+) target complex I component NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 was not affected by dopamine together with 26 other proteins. The comparison of proteomics approaches highlighted the fragmentation of some mitochondrial proteins, suggesting an alteration of the mitochondrial protease activity. Pathway and disease association analysis of the proteins affected by dopamine revealed the overrepresentation of the Parkinson's disease and the parkin-ubiquitin proteasomal system pathways and of gene ontologies associated with generation of precursor metabolites and energy, response to topologically incorrect proteins and programmed cell death. These alterations may be globally interpreted in part as the result of a direct effect of dopamine on mitochondria (e.g. alteration of the mitochondrial protease activity) and in part as the effect on mitochondria of a general activation of cellular processes (e.g. regulation of programmed cell death).

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca 2+ /calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein (GFAP)-expressing astrocytes (astroglial calcineurin knock-out, ACN-KO). Here we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs).This impairment was associated with a decrease in after-hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K + homeostasis. Indeed, blockade of Na + /K + -ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca 2+ -sensitive switch.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. PD mostly occurs sporadically and its cause remains unknown, nevertheless the discovery of familiar forms of PD, characterized by mutations of genes encoding proteins associated with mitochondria homeostasis, suggests a strong implication of the mitochondrial quality control system in PD. We investigated the effect of dopamine cytosolic accumulation in undifferentiated SH-SY5Y cells, an in vitro model widely used to reproduce impairment of dopamine homeostasis, an early step in PD pathogenesis. A strong depolarization of the mitochondrial membrane was observed after dopamine exposure. Nevertheless, mitochondrial network resulted to assume a peculiar morphology with a distinct pattern of OPA1 and MFN1, key regulators of mitochondrial dynamics. Moreover, selective elimination of dysfunctional mitochondria did not take place, suggesting an impairment of the mitophagic machinery induced by dopamine. Indeed, PINK1 did not accumulate on the outer mitochondrial membrane, nor was parkin recruited to depolarized mitochondria. Altogether, our results indicate that an improper handling of dysfunctional mitochondria may be a leading event in PD pathogenesis.

Parkinson's disease (PD) is a complex neurodegenerative disease whose etiology has not been completely characterized. Many cellular processes have been proposed to play a role in the neuronal damage and loss: defects in the proteosomal activity, altered protein processing, increased reactive oxygen species burden. Among them, the involvement of a decreased activity and an altered disposal of mitochondria is becoming more and more evident. The mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I, has been widely used to reproduce biochemical alterations linked to PD in vitro and its precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), to induce a Parkinson-like syndrome in vivo. Therefore, we performed a meta-analysis of the literature of all the proteomic investigations of neuronal alterations due to MPP+ treatment and compared it with our results obtained with a mitochondrial proteomic analysis of SH-SY5Y cells treated with MPP+. By using open-source bioinformatics tools, we identified the biochemical pathways and the molecular functions mostly affected by MPP+, i.e., ATP production, the mitochondrial unfolded stress response, apoptosis, autophagy, and, most importantly, the synapse funcionality. Eventually, we generated protein networks, based on physical or functional interactions, to highlight the relationships among the molecular actors involved. In particular, we identified the mitochondrial protein HSP60 as the central hub in the protein-protein interaction network. As a whole, this analysis clarified the cellular responses to MPP+, the specific mitochondrial proteome alterations induced and how this toxic model can recapitulate some pathogenetic events of PD.

Neurogenesis persists in the subgranular zone of the hippocampal formation in the adult mammalian brain. In this area, neural progenitor cells (NPCs) receive both permissive and instructive signals, including neurotransmitters, that allow them to generate adult-born neurons which can be functionally integrated in the preexisting circuit. Deregulation of adult hippocampal neurogenesis (ahNG) occurs in several neuropsychiatric and neurodegenerative diseases, including major depression, and represents a potential therapeutic target. Of interest, several studies suggested that, both in rodents and in humans, ahNG is increased by chronic administration of classical monoaminergic antidepressant drugs, suggesting that modulation of this process may participate to their therapeutic effects. Since the established observation that noradrenergic innervations from locus coeruleus make contact with NPC in the dentate gyrus, we investigated the role of beta adrenergic receptor (β-AR) on ahNG both in vitro and in vivo. Here we report that, in vitro, activation of β2-AR by norepinephrine and β2-AR agonists promotes the formation of NPC-derived mature neurons, without affecting NPC survival or differentiation toward glial lineages. Additionally, we show that a selective β2-AR agonist able to cross the blood–brain barrier, salmeterol, positively modulates hippocampal neuroplasticity when chronically administered in adult naïve mice. Indeed, salmeterol significantly increased number, maturation, and dendritic complexity of DCX+ neuroblasts. The increased number of DCX+ cells was not accompanied by a parallel increase in the percentage of BrdU+/DCX+ cells suggesting a potential prosurvival effect of the drug on neuroblasts. More importantly, compared to vehicle, salmeterol promoted ahNG, as demonstrated by an increase in the actual number of BrdU+/NeuN+ cells and in the percentage of BrdU+/NeuN+ cells over the total number of newly generated cells. Interestingly, salmeterol proneurogenic effects were restricted to the ventral hippocampus, an area related to emotional behavior and mood regulation. Since salmeterol is commonly used for asthma therapy in the clinical setting, its novel pharmacological property deserves to be further exploited with a particular focus on drug potential to counteract stress-induced deregulation of ahNG and depressive-like behavior.

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Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca 2+ /calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein (GFAP)-expressing astrocytes (astroglial calcineurin knock-out, ACN-KO). Here we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs).This impairment was associated with a decrease in after-hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K + homeostasis. Indeed, blockade of Na + /K + -ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca 2+ -sensitive switch.

Adolescence represents a crucial period for maturation of brain structures involved in cognition. Early in life unhealthy dietary patterns are associated with inferior cognitive outcomes at later ages; conversely, healthy diet is associated with better cognitive results. In this study we analyzed the effects of a short period of hypercaloric diet on newborn hippocampal doublecortin+ (DCX) immature neurons in adolescent mice. Male mice received high fat diet (HFD) or control low fat diet (LFD) from the 5th week of age for 1 or 2 weeks, or 1 week HFD followed by 1 week LFD. After diet supply, mice were either perfused for immunohistochemical (IHC) analysis or their hippocampi were dissected for biochemical assays. Detailed morphometric analysis was performed in DCX+ cells that displayed features of immature neurons. We report that 1 week-HFD was sufficient to dramatically reduce dendritic tree complexity of DCX+ cells. This effect occurred specifically in dorsal and not ventral hippocampus and correlated with reduced BDNF expression levels in dorsal hippocampus. Both structural and biochemical changes were reversed by a return to LFD. Altogether these studies increase our current knowledge on potential consequences of hypercaloric diet on brain and in particular on dorsal hippocampal neuroplasticity.