Open Access
Striatal synaptic bioenergetic and autophagic decline in premotor experimental parkinsonism
(Oxford University Press, 2022) Merino Galán, Leyre; Jiménez Urbieta, Haritz; Zamarbide, Marta; Rodríguez Chinchilla, Tatiana; Belloso Iguerategui, Arantzazu; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Aiastui, Ana; Doudnikoff, Evelyne.; Bézard, Erwan; Ouro, Alberto; Knafo, Shira; Gago, Belén; Quiroga Varela, Ana; Rodríguez Oroz, María Cruz; Ciencias de la Salud; Osasun Zientziak
Synaptic impairment might precede neuronal degeneration in Parkinson’s disease. However, the intimate mechanisms altering synaptic function by the accumulation of presynaptic α-synuclein in striatal dopaminergic terminals before dopaminergic death occurs, have not been elucidated. Our aim is to unravel the sequence of synaptic functional and structural changes preceding symptomatic dopaminergic cell death. As such, we evaluated the temporal sequence of functional and structural changes at striatal synapses before parkinsonian motor features appear in a rat model of progressive dopaminergic death induced by overexpression of the human mutated A53T α-synuclein in the substantia nigra pars compacta, a protein transported to these synapses. Sequential window acquisition of all theoretical mass spectra proteomics identified deregulated proteins involved first in energy metabolism and later, in vesicle cycling and autophagy. After protein deregulation and when α-synuclein accumulated at striatal synapses, alterations to mitochondrial bioenergetics were observed using a Seahorse XF96 analyser. Sustained dysfunctional mitochondrial bioenergetics was followed by a decrease in the number of dopaminergic terminals, morphological and ultrastructural alterations, and an abnormal accumulation of autophagic/endocytic vesicles inside the remaining dopaminergic fibres was evident by electron microscopy. The total mitochondrial population remained unchanged whereas the number of ultrastructurally damaged mitochondria increases as the pathological process evolved. We also observed ultrastructural signs of plasticity within glutamatergic synapses before the expression of motor abnormalities, such as a reduction in axospinous synapses and an increase in perforated postsynaptic densities. Overall, we found that a synaptic energetic failure and accumulation of dysfunctional organelles occur sequentially at the dopaminergic terminals as the earliest events preceding structural changes and cell death. We also identify key proteins involved in these earliest functional abnormalities that may be modulated and serve as therapeutic targets to counterbalance the degeneration of dopaminergic cells to delay or prevent the development of Parkinson’s disease.
Open Access
Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models
(Elsevier, 2023) Vinueza-Gavilanes, Rodrigo; Bravo-González, Jorge Juan; Basurco, Leyre; Boncristiani, Chiara; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Marcilla, Irene; Pérez Mediavilla, Alberto; Luquin, María Rosario; Vales, Africa; González-Aseguinolaza, Gloria; Aymerich, María Soledad; Aragón, Tomás; Arrasate, Montserrat; Ciencias de la Salud; Osasun Zientziak
Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14‐3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14‐3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14‐3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.
Open Access
Alteration in the cerebrospinal fluid lipidome in Parkinson’s disease: a post-mortem pilot study
(MDPI, 2021) Fernández Irigoyen, Joaquín; Cartas Cejudo, Paz; Iruarrizaga-Lejarreta, Marta; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak
Lipid metabolism is clearly associated to Parkinson’s disease (PD). Although lipid homeostasis has been widely studied in multiple animal and cellular models, as well as in blood derived from PD individuals, the cerebrospinal fluid (CSF) lipidomic profile in PD remains largely unexplored. In this study, we characterized the post-mortem CSF lipidomic imbalance between neurologically intact controls (n = 10) and PD subjects (n = 20). The combination of dual extraction with ultra-performance liquid chromatography-electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC-ESI-qToF-MS/MS) allowed for the monitoring of 257 lipid species across all samples. Complementary multivariate and univariate data analysis identified that glycerolipids (mono-, di-, and triacylglycerides), saturated and mono/polyunsaturated fatty acids, primary fatty amides, glycerophospholipids (phosphatidylcholines, phosphatidylethanolamines), sphingolipids (ceramides, sphingomyelins), N-acylethanolamines and sterol lipids (cholesteryl esters, steroids) were significantly increased in the CSF of PD compared to the control group. Interestingly, CSF lipid dyshomeostasis differed depending on neuropathological staging and disease duration. These results, despite the limitation of being obtained in a small population, suggest extensive CSF lipid remodeling in PD, shedding new light on the deployment of CSF lipidomics as a promising tool to identify potential lipid markers as well as discriminatory lipid species between PD and other atypical parkinsonisms.
Open Access
Hippocampal synaptic failure is an early event in experimental parkinsonism with subtle cognitive deficit
(Oxford University Press, 2023) Belloso Iguerategui, Arantzazu; Zamarbide, Marta; Merino Galán, Leyre; Rodríguez Chinchilla, Tatiana; Gago, Belén; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Cotman, Carl W.; Prieto, G. Aleph; Quiroga Varela, Ana; Rodríguez Oroz, María Cruz; Ciencias de la Salud; Osasun Zientziak
Learning and memory mainly rely on correct synaptic function in the hippocampus and other brain regions. In Parkinson’s disease, subtle cognitive deficits may even precede motor signs early in the disease. Hence, we set out to unravel the earliest hippocampal synaptic alterations associated with human α-synuclein overexpression prior to and soon after the appearance of cognitive deficits in a parkinsonism model. We bilaterally injected adeno-associated viral vectors encoding A53T-mutated human α-synuclein into the substantia nigra of rats, and evaluated them 1, 2, 4 and 16 weeks post-inoculation by immunohistochemistry and immunofluorescence to study degeneration and distribution of α-synuclein in the midbrain and hippocampus. The object location test was used to evaluate hippocampal-dependent memory. Sequential window acquisition of all theoretical mass spectrometry-based proteomics and fluorescence analysis of single-synapse long-term potentiation were used to study alterations to protein composition and plasticity in isolated hippocampal synapses. The effect of L-DOPA and pramipexole on long-term potentiation was also tested. Human α-synuclein was found within dopaminergic and glutamatergic neurons of the ventral tegmental area, and in dopaminergic, glutamatergic and GABAergic axon terminals in the hippocampus from 1 week post-inoculation, concomitant with mild dopaminergic degeneration in the ventral tegmental area. In the hippocampus, differential expression of proteins involved in synaptic vesicle cycling, neurotransmitter release and receptor trafficking, together with impaired long-term potentiation were the first events observed (1 week post-inoculation), preceding cognitive deficits (4 weeks post-inoculation). Later on, at 16 weeks post-inoculation, there was a deregulation of proteins involved in synaptic function, particularly those involved in the regulation of membrane potential, ion balance and receptor signalling. Hippocampal long-term potentiation was impaired before and soon after the onset of cognitive deficits, at 1 and 4 weeks post-inoculation, respectively. L-DOPA recovered hippocampal long-term potentiation more efficiently at 4 weeks post-inoculation than pramipexole, which partially rescued it at both time points. Overall, we found impaired synaptic plasticity and proteome dysregulation at hippocampal terminals to be the first events that contribute to the development of cognitive deficits in experimental parkinsonism. Our results not only point to dopaminergic but also to glutamatergic and GABAergic dysfunction, highlighting the relevance of the three neurotransmitter systems in the ventral tegmental area-hippocampus interaction from the earliest stages of parkinsonism. The proteins identified in the current work may constitute potential biomarkers of early synaptic damage in the hippocampus and hence, therapies targeting these could potentially restore early synaptic malfunction and consequently, cognitive deficits in Parkinson’s disease.
Open Access
In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson's disease
(Oxford University Press, 2024-12-20) Cortés, Adriana; Phung, Toan K.; Mena, Lorena de ; Garrido, Alicia; Infante, Jon; Ruíz-Martínez, Javier; Galmés-Ordinas, Miquel À.; Glendinning, Sophie; Pérez, Jesica ; Roig, Ana ; Soto, Marta; Cosgaya, Marina; Ravasi, Valeria; Fernández, Manel; Rubiano-Castro, Alejandro ; Díaz, Ramón; Hernández-Eguiazu, Haizea ; Sánchez-Quintana, Coro; Vinagre-Aragón, Ana; Mondragón, Elisabet; Croitoru, Ioana; Rivera-Sánchez, María ; Corrales-Pardo, Andrea; Sierra, María; Tolosa, Eduardo; Malagelada, Cristina; Nirujogi, Raja S.; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Alessi, Dario R.; Martí, María J.; Ezquerra, Mario; Fernández-Santiago, Rubén; Ciencias de la Salud; Osasun Zientziak
Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson's disease (L2PD) and idiopathic PD. However, pharmacodynamic readouts and progression biomarkers for clinical trials aiming for disease modification are insufficient, given that no endogenous marker reflecting enhanced kinase activity of the most common LRRK2 G2019S mutation has yet been reported in L2PD patients.Using phospho-/proteomic analyses, we assessed the impact of LRRK2-activating mutations in peripheral blood mononuclear cells from an LRRK2 clinical cohort from Spain (n = 174). The study groups encompassed G2019S L2PD patients (n = 37), non-manifesting LRRK2 mutation carriers of G2019S (here termed G2019S L2NMCs) (n = 27), R1441G L2PD patients (n = 14), R1441G L2NMCs (n = 11), idiopathic PD patients (n = 40) and healthy controls (n = 45).We identified 207 differentially regulated proteins in G2019S L2PD compared with controls (39 upregulated and 168 downregulated) and 67 in G2019S L2NMCs (10 upregulated and 57 downregulated). G2019S downregulated proteins affected the endolysosomal pathway, proteostasis and mitochondria, e.g. ATIC, RAB9A or LAMP1. At the phospho-proteome level, we observed increases in endogenous phosphorylation levels of pSer106 RAB12 in G2019S carriers, which were validated by immunoblotting after 1 year of follow-up (n = 48). Freshly collected peripheral blood mononuclear cells from three G2019S L2PD, one R1441G L2PD, one idiopathic PD and five controls (n = 10) showed strong diminishment of pSer106 RAB12 phosphorylation levels after in vitro administration of the MLi-2 LRRK2 inhibitor. Using machine learning, we identified an 18-feature G2019S phospho-/protein signature discriminating G2019S L2PD, L2NMCs and controls with 96% accuracy that was correlated with disease severity, i.e. UPDRS-III motor scoring.Using easily accessible peripheral blood mononuclear cells from a LRRK2 clinical cohort, we identified elevated levels of pSer106 RAB12 as an endogenous biomarker of G2019S carriers. Our data suggest that monitoring pSer106 RAB12 phosphorylation could be a relevant biomarker for tracking LRRK2 activation, particularly in G2019S carriers. Future work might determine whether pSer106 RAB12 could help with patient enrichment and monitoring drug efficacy in LRRK2 clinical trials. The LRRK2 activating mutation G2019S is the most frequent genetic cause of Parkinson's disease. Through phospho-proteome analysis of blood, Cort & eacute;s et al. identify elevated phospho-RAB12 levels as an endogenous biomarker of G2019S mutation carriers, with potential utility in clinical trials.