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

Brain tyrosinase; Neuromelanin production; Parkinson’sTirosinasa cerebral; Producció de neuromelanina; ParkinsonTirosinasa cerebral; Producción de neuromelanina; ParkinsonIn 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

Transcriptomic changes linked to age-dependent neuromelanin accumulation in a new Parkinson's disease mouse model

Resumen del trabajo presentado en el 50th Annual Meeting Society for Neuroscience, celebrado de forma virtual del 8 al 11 de noviembre de 2021In Parkinson's disease (PD) there is a preferential degeneration of neuromelanin (NM)-containing neurons, especially neurons from the Substantia Nigra (SN) but also from the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC). We generated a new NM-producing mouse model, based on the tissue-specific constitutive expression of human tyrosinase (hTyr) under the tyrosine hydroxylase (TH) promoter (tgNM), that mimics the distribution and age-dependent accumulation of NM in the human brain (i.e. catecholaminergic groups A1-A14). TgNM mice exhibited major PD features, including both motor and non-motor behavioral alterations, inclusion body formation, neuronal degeneration in lower brainstem areas (LC) together with neuronal dysfunction in higher brainstem areas (SN and VTA). In order to understand the mechanisms by which NM accumulation in specific brain areas ultimately interferes with the normal functioning of cells, we characterized genome-wide transcriptomic changes linked to the intracellular presence and progressive accumulation of NM in two NM-accumulating neuronal subpopulations (SN and VTA) that are known to be differentially susceptible to PD pathology. We selectively isolated single dopaminergic NM-containing neurons by laser capture microdissection from male and female wild-type and tgNM animals at 3 months, 12 months and 20 months of age (n=4-6 mice per group). We performed differential expression analysis, resulting in statistically significant differentially expressed genes at all ages (p-value<0.5). Gene-set enrichment analysis (GSEA) with Reactome Pathway Database led to the identification of altered biological pathways in tgNM related to neuroinflammation, vesicle-mediated transport and lipid metabolism, transcription and translation, mitochondrial function and cell cycle (senescence) (False Discovery Rate<0.05). Targeted-based validation of candidate RNA species was performed in microdissected samples by quantitative real-time PCR and candidate biological pathways were validated at the protein level by western blot in dissected ventral midbrain tissues from biological replicates. The transcriptomic profiles identified in this project contribute to our understanding of selective vulnerability in PD and brain aging, and points to key biological pathways and molecular targets in prodromal and early PD

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

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

Abstract 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

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

Abstract 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

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. Neurons that accumulate the highest levels of neuromelanin with age are also the most susceptible to degeneration in Parkinson's disease. Using a neuromelanin-producing rat model, Gonzalez-Sepulveda, Compte et al. show that slowing down age-dependent neuromelanin production can help prevent Parkinson's disease-like features

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