Adult-specific Reelin expression alters striatal neuronal organization: implications for neuropsychiatric disorders.

In addition to neuronal migration, brain development, and adult plasticity, the extracellular matrix protein Reelin has been extensively implicated in human psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. Moreover, heterozygous reeler mice exhibit features reminiscent of these disorders, while overexpression of Reelin protects against its manifestation. However, how Reelin influences the structure and circuits of the striatal complex, a key region for the above-mentioned disorders, is far from being understood, especially when altered Reelin expression levels are found at adult stages. In the present study, we took advantage of complementary conditional gain- and loss-of-function mouse models to investigate how Reelin levels may modify adult brain striatal structure and neuronal composition. Using immunohistochemical techniques, we determined that Reelin does not seem to influence the striatal patch and matrix organization (studied by µ-opioid receptor immunohistochemistry) nor the density of medium spiny neurons (MSNs, studied with DARPP-32). We show that overexpression of Reelin leads to increased numbers of striatal parvalbumin- and cholinergic-interneurons, and to a slight increase in tyrosine hydroxylase-positive projections. We conclude that increased Reelin levels might modulate the numbers of striatal interneurons and the density of the nigrostriatal dopaminergic projections, suggesting that these changes may be involved in the protection of Reelin against neuropsychiatric disorders.

In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson's disease.

Humans accumulate with age the dark-brown pigment neuromelanin inside specific neuronal groups. Neurons with the highest neuromelanin levels are particularly susceptible to degeneration in Parkinson's disease, especially dopaminergic neurons of the substantia nigra, the loss of which leads to characteristic motor Parkinson's disease symptoms. In contrast to humans, neuromelanin does not appear spontaneously in most animals, including rodents, and Parkinson's disease is an exclusively human condition. Using humanized neuromelanin-producing rodents, we recently found that neuromelanin can trigger Parkinson's disease pathology when accumulated above a specific pathogenic threshold. Here, by taking advantage of this newly developed animal model, we assessed whether the intracellular build-up of neuromelanin that occurs with age can be slowed down in vivo to prevent or attenuate Parkinson's disease. Because neuromelanin derives from the oxidation of free cytosolic dopamine, we enhanced dopamine vesicular encapsulation in the substantia nigra of neuromelanin-producing rats by viral vector-mediated overexpression of vesicular monoamine transporter 2 (VMAT2). This strategy reduced the formation of potentially toxic oxidized dopamine species that can convert into neuromelanin and maintained intracellular neuromelanin levels below their pathogenic threshold. Decreased neuromelanin production was associated with an attenuation of Lewy body-like inclusion formation and a long-term preservation of dopamine homeostasis, nigrostriatal neuronal integrity and motor function in these animals. Our results demonstrate the feasibility and therapeutic potential of modulating age-dependent intracellular neuromelanin production in vivo, thereby opening an unexplored path for the treatment of Parkinson's disease and, in a broader sense, brain ageing.

Validation of a Reversed Phase UPLC-MS/MS Method to Determine Dopamine Metabolites and Oxidation Intermediates in Neuronal Differentiated SH-SY5Y Cells and Brain Tissue.

Dopamine is a key neurotransmitter in the pathophysiology of various neurological disorders such as addiction or Parkinson's disease. Disturbances in its metabolism could lead to dopamine accumulation in the cytoplasm and an increased production of o-quinones and their derivatives, which have neurotoxic potential and act as precursors in neuromelanin synthesis. Thus, quantification of the dopaminergic metabolism is essential for monitoring changes that may contribute to disease development. Here, we developed and validated an UPLC-MS/MS method to detect and quantify a panel of eight dopaminergic metabolites, including the oxidation product aminochrome. Our method was validated in differentiated SH-SY5Y cells and mouse brain tissue and was then employed in brain samples from humans and rats to ensure method reliability in different matrices. Finally, to prove the biological relevance of our method, we determined metabolic changes in an in vitro cellular model of dopamine oxidation/neuromelanin production and in human postmortem samples from Parkinson's disease patients. The current study provides a validated method to simultaneously monitor possible alterations in dopamine degradation and o-quinone production pathways that can be applied to in vitro and in vivo experimental models of neurological disorders and human brain samples.

Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson's disease pathogenesis.

In Parkinson's disease (PD) there is a selective degeneration of neuromelanin-containing neurons, especially substantia nigra dopaminergic neurons. In humans, neuromelanin accumulates with age, the latter being the main risk factor for PD. The contribution of neuromelanin to PD pathogenesis remains unknown because, unlike humans, common laboratory animals lack neuromelanin. Synthesis of peripheral melanins is mediated by tyrosinase, an enzyme also present at low levels in the brain. Here we report that overexpression of human tyrosinase in rat substantia nigra results in age-dependent production of human-like neuromelanin within nigral dopaminergic neurons, up to levels reached in elderly humans. In these animals, intracellular neuromelanin accumulation above a specific threshold is associated to an age-dependent PD phenotype, including hypokinesia, Lewy body-like formation and nigrostriatal neurodegeneration. Enhancing lysosomal proteostasis reduces intracellular neuromelanin and prevents neurodegeneration in tyrosinase-overexpressing animals. Our results suggest that intracellular neuromelanin levels may set the threshold for the initiation of PD.

Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson's disease.

Mitochondria are the prime energy source in most eukaryotic cells, but these highly dynamic organelles are also involved in a multitude of cellular events. Disruption of mitochondrial homeostasis and the subsequent mitochondrial dysfunction plays a key role in the pathophysiology of Parkinson's disease (PD). Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for neuronal survival. Here, we have studied the mitochondrial protein import system in in vitro and in vivo models of PD. Complex I inhibition, a characteristic pathological hallmark in PD, impaired mitochondrial protein import, which was associated with a downregulation of two key components of the system: translocase of the outer membrane 20 (TOM20) and translocase of the inner membrane 23 (TIM23), both in vitro and in vivo. In vitro, those changes were associated with OXPHOS protein downregulation, accumulation of aggregated proteins inside mitochondria and downregulation of mitochondrial chaperones. Most of these pathogenic changes, including mitochondrial dysfunction and dopaminergic cell death, were abrogated by TOM20 or TIM23 overexpression, in vitro. However, in vivo, while TOM20 overexpression exacerbated neurodegeneration in both substantia nigra (SN) pars compacta (pc) and striatum, overexpression of TIM23 partially protected dopaminergic neurons in the SNpc. These results highlight mitochondrial protein import dysfunction and the distinct role of two of their components in the pathogenesis of PD and suggest the need for future studies to further characterize mitochondrial protein import deficit in the context of PD.

Overexpression of TFEB Drives a Pleiotropic Neurotrophic Effect and Prevents Parkinson's Disease-Related Neurodegeneration.

The possible implication of transcription factor EB (TFEB) as a therapeutic target in Parkinson's disease has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the lysosomal-autophagic system. Here, we assessed the effect of overexpressing TFEB with an adeno-associated viral vector in mouse substantia nigra dopaminergic neurons. We demonstrate that TFEB overexpression drives a previously unknown bona fide neurotrophic effect, giving rise to cell growth, higher tyrosine hydroxylase levels, and increased dopamine release in the striatum. TFEB overexpression induces the activation of the mitogen-activated protein kinase 1/3 (MAPK1/3) and AKT pro-survival pathways, phosphorylation of mTORC1 effectors 4E-binding protein 1 (4E-BP1) and S6 kinase B1 (S6K1), and increased protein synthesis. We show that TFEB overexpression prevents dopaminergic cell loss and counteracts atrophy and the associated protein synthesis decline in the MPTP mouse model of Parkinson's disease. Our results suggest that increasing TFEB activity might prevent neuronal death and restore neuronal function in Parkinson's disease and other neurodegenerative diseases through different mechanisms.

Lack of pathogenic potential of peripheral α-synuclein aggregates from Parkinson's disease patients.

In Parkinson's disease (PD) there is widespread accumulation in the brain of abnormal α-synuclein aggregates forming intraneuronal Lewy bodies (LB). It is now well established that LB-type α-synuclein aggregates also occur in the peripheral autonomic nervous system in PD, from where it has been speculated they may progressively spread to the central nervous system through synaptically-connected brain networks and reach the substantia nigra to trigger herein dopaminergic dysfunction/degeneration and subsequent parkinsonism. Supporting a pathogenic role for α-synuclein aggregates we have previously shown that LB purified from postmortem PD brains promote α-synuclein pathology and dopaminergic neurodegeneration when intracerebrally inoculated into wild-type mice. However, the pathogenic capacity of PD-derived peripheral α-synuclein aggregates remains unknown. Here we addressed this question using purified LB-type α-synuclein aggregates from postmortem PD stellate ganglia (SG), a paravertebral sympathetic ganglion that exhibits consistent and conspicuous Lewy pathology in all PD patients. In contrast to our previous findings using nigral LB extracts, intracerebral inoculation of SG-derived LB into mice did not trigger long-term nigrostriatal neurodegeneration nor α-synuclein pathology. The differential pathogenic capacities of central- and peripheral-derived α-synuclein aggregates appear independent of the absolute amount and basic biochemical properties of α-synuclein within these aggregates and may rely instead on differences in α-synuclein conformation and/or yet unrecognized brain region-specific intrinsic factors. Our results argue against a putative pathogenic capacity of peripheral α-synuclein aggregates to promote α-synuclein pathology in the brain, propagate between neuronal networks or induce neurodegeneration.

Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys.

OBJECTIVE: Mounting evidence suggests that α-synuclein, a major protein component of Lewy bodies (LB), may be responsible for initiating and spreading the pathological process in Parkinson disease (PD). Supporting this concept, intracerebral inoculation of synthetic recombinant α-synuclein fibrils can trigger α-synuclein pathology in mice. However, it remains uncertain whether the pathogenic effects of recombinant synthetic α-synuclein may apply to PD-linked pathological α-synuclein and occur in species closer to humans. METHODS: Nigral LB-enriched fractions containing pathological α-synuclein were purified from postmortem PD brains by sucrose gradient fractionation and subsequently inoculated into the substantia nigra or striatum of wild-type mice and macaque monkeys. Control animals received non-LB fractions containing soluble α-synuclein derived from the same nigral PD tissue. RESULTS: In both mice and monkeys, intranigral or intrastriatal inoculations of PD-derived LB extracts resulted in progressive nigrostriatal neurodegeneration starting at striatal dopaminergic terminals. No neurodegeneration was observed in animals receiving non-LB fractions from the same patients. In LB-injected animals, exogenous human α-synuclein was quickly internalized within host neurons and triggered the pathological conversion of endogenous α-synuclein. At the onset of LB-induced degeneration, host pathological α-synuclein diffusely accumulated within nigral neurons and anatomically interconnected regions, both anterogradely and retrogradely. LB-induced pathogenic effects required both human α-synuclein present in LB extracts and host expression of α-synuclein. INTERPRETATION: α-Synuclein species contained in PD-derived LB are pathogenic and have the capacity to initiate a PD-like pathological process, including intracellular and presynaptic accumulations of pathological α-synuclein in different brain areas and slowly progressive axon-initiated dopaminergic nigrostriatal neurodegeneration.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice.

Monocarboxylate transporters (MCTs) are membrane carriers for lactate and ketone bodies. Three isoforms, MCT1, MCT2 and MCT4, have been described in the central nervous system but little information is available about the regulation of their expression in relation to altered metabolic and/or nutritional conditions. We show here that brains of mice fed on a high fat diet (HFD) up to 12 weeks as well as brains of genetically obese (ob/ob) or diabetic (db/db) mice exhibit an increase of MCT1, MCT2 and MCT4 expression as compared to brains of control mice fed a standard diet. Enhanced expression of each transporter was visible throughout the brain but most prominently in the cortex and in the hippocampus. Using immunohistochemistry, we observed that neurons (expressing mainly MCT2 but also sometimes low levels of MCT1 under normal conditions) were immunolabelled for all three transporters in HFD mice as well as in ob/ob and db/db mice. At the subcellular level, changes were most remarkable in neuronal cell bodies. Western blotting performed on brain structure extracts allowed us to confirm quantitatively the enhancement of MCT1 and MCT2 expression. Our data demonstrate that the expression of cerebral MCT isoforms can be modulated by alterations of peripheral metabolism, suggesting that the adult brain is sensitive and adapts to new metabolic states. This observation could be relevant in the context of obesity development and its consequences for brain function.