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1.
Mol Cell Proteomics ; 23(2): 100708, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38154689

RESUMEN

In the era of open-modification search engines, more posttranslational modifications than ever can be detected by LC-MS/MS-based proteomics. This development can switch proteomics research into a higher gear, as PTMs are key in many cellular pathways important in cell proliferation, migration, metastasis, and aging. However, despite these advances in modification identification, statistical methods for PTM-level quantification and differential analysis have yet to catch up. This absence can partly be explained by statistical challenges inherent to the data, such as the confounding of PTM intensities with its parent protein abundance. Therefore, we have developed msqrob2PTM, a new workflow in the msqrob2 universe capable of differential abundance analysis at the PTM and at the peptidoform level. The latter is important for validating PTMs found as significantly differential. Indeed, as our method can deal with multiple PTMs per peptidoform, there is a possibility that significant PTMs stem from one significant peptidoform carrying another PTM, hinting that it might be the other PTM driving the perceived differential abundance. Our workflows can flag both differential peptidoform abundance (DPA) and differential peptidoform usage (DPU). This enables a distinction between direct assessment of differential abundance of peptidoforms (DPA) and differences in the relative usage of peptidoforms corrected for corresponding protein abundances (DPU). For DPA, we directly model the log2-transformed peptidoform intensities, while for DPU, we correct for parent protein abundance by an intermediate normalization step which calculates the log2-ratio of the peptidoform intensities to their summarized parent protein intensities. We demonstrated the utility and performance of msqrob2PTM by applying it to datasets with known ground truth, as well as to biological PTM-rich datasets. Our results show that msqrob2PTM is on par with, or surpassing the performance of, the current state-of-the-art methods. Moreover, msqrob2PTM is currently unique in providing output at the peptidoform level.


Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Cromatografía Liquida , Procesamiento Proteico-Postraduccional , Proteínas
2.
Int J Mol Sci ; 25(16)2024 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-39201743

RESUMEN

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.


Asunto(s)
Axones , Dependovirus , MicroARNs , Regeneración Nerviosa , Neuritas , MicroARNs/genética , MicroARNs/metabolismo , Animales , Axones/metabolismo , Axones/fisiología , Regeneración Nerviosa/genética , Neuritas/metabolismo , Dependovirus/genética , Células Cultivadas , Vectores Genéticos/genética , Ratones , Neuronas/metabolismo
3.
J Neurochem ; 166(5): 862-874, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37515330

RESUMEN

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non-motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD-associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post-mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late-stage PD and represents a putative target for novel therapeutic strategies.


Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Parkinson , Humanos , Persona de Mediana Edad , Enfermedad de Parkinson/metabolismo , Proteómica/métodos , Hipocampo/metabolismo , Enfermedad de Alzheimer/patología
4.
J Neurochem ; 159(3): 554-573, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34176164

RESUMEN

Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240 mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12 weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90 days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.


Asunto(s)
Química Encefálica , Hierro/farmacología , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidad , Animales , Animales Recién Nacidos , Conectoma , Cuerpo Estriado , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Hierro/administración & dosificación , Masculino , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/psicología , Ratones Endogámicos C57BL , Microglía/patología , Microinyecciones , Actividad Motora/efectos de los fármacos , alfa-Sinucleína/administración & dosificación
5.
Neurobiol Dis ; 119: 121-135, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30092270

RESUMEN

Alpha-synuclein (aSyn) is the major protein component of Lewy bodies and Lewy neurites, the typical pathological hallmarks in Parkinson's disease (PD) and Dementia with Lewy bodies. aSyn is capable of inducing transcriptional deregulation, but the precise effect of specific aSyn mutants associated with familial forms of PD, remains unclear. Here, we used transgenic mice overexpressing human wild-type (WT) or A30P aSyn to compare the transcriptional profiles of the two animal models. We found that A30P aSyn promotes strong transcriptional deregulation and increases DNA binding. Interestingly, COL4A2, a major component of basement membranes, was found to be upregulated in both A30P aSyn transgenic mice and in dopaminergic neurons expressing A30P aSyn, suggesting a crucial role for collagen related genes in aSyn-induced toxicity. Finally, we observed that A30P aSyn alters Golgi morphology and increases the susceptibility to endoplasmic reticulum (ER) stress in dopaminergic cells. In total, our findings provide novel insight into the putative role of aSyn on transcription and on the molecular mechanisms involved, thereby opening novel avenues for future therapeutic interventions in PD and other synucleinopathies.


Asunto(s)
Colágeno Tipo IV/biosíntesis , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Fragmentos de Péptidos/biosíntesis , alfa-Sinucleína/biosíntesis , Animales , Células Cultivadas , Colágeno Tipo IV/genética , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/genética , alfa-Sinucleína/genética
6.
Nat Commun ; 15(1): 4893, 2024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38849340

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.


Asunto(s)
Esclerosis Amiotrófica Lateral , Modelos Animales de Enfermedad , Sistema de Señalización de MAP Quinasas , Ratones Transgénicos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Femenino , Animales , Masculino , Ratones , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Piridonas/farmacología , Piridonas/uso terapéutico , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Corteza Prefrontal/metabolismo , Transcriptoma , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Persona de Mediana Edad , MicroARNs/genética , MicroARNs/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Caracteres Sexuales , Anciano , Factores Sexuales , Pirimidinonas
7.
Brain Pathol ; 33(5): e13196, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37485772

RESUMEN

Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) are neurodegenerative disorders with alpha-synuclein (α-syn) aggregation pathology. Different strains of α-syn with unique properties are suggested to cause distinct clinical and pathological manifestations resulting in PD, MSA, or DLB. To study individual α-syn spreading patterns, we injected α-syn fibrils amplified from brain homogenates of two MSA patients and two PD patients into the brains of C57BI6/J mice. Antibody staining against pS129-α-syn showed that α-syn fibrils amplified from the brain homogenates of the four different patients caused different levels of α-syn spreading. The strongest α-syn pathology was triggered by α-syn fibrils of one of the two MSA patients, followed by comparable pS129-α-syn induction by the second MSA and one PD patient material. Histological analysis using an antibody against Iba1 further showed that the formation of pS129-α-syn is associated with increased microglia activation. In contrast, no differences in dopaminergic neuron numbers or co-localization of α-syn in oligodendrocytes were observed between the different groups. Our data support the spreading of α-syn pathology in MSA, while at the same time pointing to spreading heterogeneity between different patients potentially driven by individual patient immanent factors.


Asunto(s)
Atrofia de Múltiples Sistemas , Enfermedad de Parkinson , Sinucleinopatías , Animales , Ratones , alfa-Sinucleína/metabolismo , Anticuerpos , Encéfalo/patología , Atrofia de Múltiples Sistemas/patología , Enfermedad de Parkinson/patología , Sinucleinopatías/patología
8.
Clin Transl Med ; 12(1): e692, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35090094

RESUMEN

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder whose prevalence is rapidly increasing worldwide. The molecular mechanisms underpinning the pathophysiology of sporadic PD remain incompletely understood. Therefore, causative therapies are still elusive. To obtain a more integrative view of disease-mediated alterations, we investigated the molecular landscape of PD in human post-mortem midbrains, a region that is highly affected during the disease process. METHODS: Tissue from 19 PD patients and 12 controls were obtained from the Parkinson's UK Brain Bank and subjected to multi-omic analyses: small and total RNA sequencing was performed on an Illumina's HiSeq4000, while proteomics experiments were performed in a hybrid triple quadrupole-time of flight mass spectrometer (TripleTOF5600+) following quantitative sequential window acquisition of all theoretical mass spectra. Differential expression analyses were performed with customized frameworks based on DESeq2 (for RNA sequencing) and with Perseus v.1.5.6.0 (for proteomics). Custom pipelines in R were used for integrative studies. RESULTS: Our analyses revealed multiple deregulated molecular targets linked to known disease mechanisms in PD as well as to novel processes. We have identified and experimentally validated (quantitative real-time polymerase chain reaction/western blotting) several PD-deregulated molecular candidates, including miR-539-3p, miR-376a-5p, miR-218-5p and miR-369-3p, the valid miRNA-mRNA interacting pairs miR-218-5p/RAB6C and miR-369-3p/GTF2H3, as well as multiple proteins, such as CHI3L1, HSPA1B, FNIP2 and TH. Vertical integration of multi-omic analyses allowed validating disease-mediated alterations across different molecular layers. Next to the identification of individual molecular targets in all explored omics layers, functional annotation of differentially expressed molecules showed an enrichment of pathways related to neuroinflammation, mitochondrial dysfunction and defects in synaptic function. CONCLUSIONS: This comprehensive assessment of PD-affected and control human midbrains revealed multiple molecular targets and networks that are relevant to the disease mechanism of advanced PD. The integrative analyses of multiple omics layers underscore the importance of neuroinflammation, immune response activation, mitochondrial and synaptic dysfunction as putative therapeutic targets for advanced PD.


Asunto(s)
Mesencéfalo/patología , Terapia Molecular Dirigida/métodos , Enfermedad de Parkinson/terapia , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Mesencéfalo/anatomía & histología , Mesencéfalo/efectos de los fármacos , Persona de Mediana Edad , Terapia Molecular Dirigida/estadística & datos numéricos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/mortalidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/estadística & datos numéricos , Reino Unido
9.
Cell Death Differ ; 27(10): 2810-2827, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32341448

RESUMEN

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.


Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia , Axones , Sistema Nervioso Central , Degeneración Nerviosa , Enfermedades Neurodegenerativas , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Homólogo de la Proteína 1 Relacionada con la Autofagia/fisiología , Axones/metabolismo , Axones/patología , Células Cultivadas , Sistema Nervioso Central/lesiones , Sistema Nervioso Central/metabolismo , Femenino , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Cultivo Primario de Células , Ratas
10.
Front Neurosci ; 12: 625, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30233304

RESUMEN

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Its main neuropathological hallmarks are the degeneration of dopaminergic neurons in the substantia nigra and alpha-synuclein containing protein inclusions, called Lewy Bodies. The diagnosis of idiopathic PD is still based on the assessment of clinical criteria, leading to an insufficient diagnostic accuracy. Additionally, there is no biomarker available allowing the prediction of the disease course or monitoring the response to therapeutic approaches. So far, protein biomarker candidates such as alpha-synuclein have failed to improve diagnosis of PD. Circulating microRNAs (miRNAs) in body fluids are promising biomarker candidates for PD, as they are easily accessible by non- or minimally-invasive procedures and changes in their expression are associated with pathophysiological processes relevant for PD. Advances in miRNA analysis methods resulted in numerous recent publications on miRNAs as putative biomarkers. Here, we discuss the applicability of different body fluids as sources for miRNA biomarkers, highlight technical aspects of miRNA analysis and give an overview on published studies investigating circulating miRNAs as biomarker candidates for diagnosis of PD and other Parkinsonian syndromes.

11.
Mol Ther Nucleic Acids ; 11: 9-22, 2018 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-29858093

RESUMEN

Parkinson's disease (PD) is the second-most-frequent neurodegenerative disorder worldwide. One major hallmark of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra. Glial cell line-derived neurotrophic factor (GDNF) potently increases DA neuron survival in models of PD; however, the underlying mechanisms are incompletely understood. MicroRNAs (miRNAs) are small, non-coding RNAs that are important for post-transcriptional regulation of gene expression. Using small RNA sequencing, we show that GDNF specifically increases the expression of miR-182-5p and miR-183-5p in primary midbrain neurons (PMNs). Transfection of synthetic miR-182-5p and miR-183-5p mimics leads to increased neurite outgrowth and mediates neuroprotection of DA neurons in vitro and in vivo, mimicking GDNF effects. This is accompanied by decreased expression of FOXO3 and FOXO1 transcription factors and increased PI3K-Akt signaling. Inhibition of endogenous miR-182-5p or miR-183-5p in GDNF-treated PMNs attenuated the pro-DA effects of GDNF. These findings unveil an unknown miR-mediated mechanism of GDNF action and suggest that targeting miRNAs is a new therapeutic avenue to PD phenotypes.

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