Dopaminergic neurons (DA) of the substantia nigra
pars compacta
(SN
pc
) selectively and progressively degenerate in Parkinson’s disease (PD). Until now, molecular analyses of DA in PD have been ...limited to genomic or transcriptomic approaches, whereas, to the best of our knowledge, no proteomic or combined multiomic study examining the protein profile of these neurons is currently available. In this exploratory study, we used laser capture microdissection to extract regions from DA in 10 human SN
pc
obtained at autopsy in PD patients and control subjects. Extracted RNA and proteins were identified by RNA sequencing and nanoliquid chromatography–mass spectrometry, respectively, and the differential expression between PD and control group was assessed. Qualitative analyses confirmed that the microdissection protocol preserves the integrity of our samples and offers access to specific molecular pathways. This multiomic analysis highlighted differential expression of 52 genes and 33 proteins, including molecules of interest already known to be dysregulated in PD, such as
LRP2
,
PNMT
,
CXCR4
,
MAOA
and
CBLN1
genes, or the Aldehyde dehydrogenase 1 protein. On the other hand, despite the same samples were used for both analyses, correlation between RNA and protein expression was low, as exemplified by the
CST3
gene encoding for the cystatin C protein. This is the first exploratory study analyzing both gene and protein expression of laser-dissected neuronal parts from SN
pc
in PD. Data are available via ProteomeXchange with identifier PXD024748 and via GEO with identifier GSE 169755.
Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about ...protein modifications in Ph− MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph− MPN pathogenesis.
•Ph-MPN erythrocytes present proteome abnormalities.•IQGAP1 is overexpressed in MPN erythrocytes.•IQGAP1/Rho GTPase signaling is deregulated in MPN erythrocytes.•IQGAP1/Rho GTPase deregulation depends on the genetic status (JAK2/CALR).
Purpose:
In preclinical studies, the Rapid‐Steady‐State‐T1 (RSST1) MRI method has advantages over conventional MRI methods for blood volume fraction (BVf) mapping, since after contrast agent ...administration, the BVf is directly quantifiable from the signal amplitude corresponding to the vascular equilibrium magnetization. This study focuses on its clinical implementation and feasibility.
Methods:
Following sequence implementation on clinical Philips Achieva scanners, the RSST1‐method is assessed at 1.5 and 3 T in the follow‐up examination of neurooncological patients receiving 0.1–0.2 mmol/kg Gd‐DOTA to determine the threshold dose needed for cerebral BVf quantification. Confounding effects on BVf quantification such as transendothelial water exchange, transverse relaxation, and contrast agent extravasation are evaluated.
Results:
For a dose ≥0.13 mmol/kg at 1.5 T and ≥0.16 mmol/kg at 3 T, the RSST1‐signal time course in macrovessels and brain tissue with Gd‐DOTA impermeable vasculature reaches a steady state at maximum amplitude for about 8 s. In macrovessels, a BVf of 100% was obtained validating cerebral microvascular BVf quantification (3.5%–4.5% in gray matter and 1.5%–2.0% in white matter). In tumor tissue, a continuously increasing signal is detected, necessitating signal modeling for tumor BVf calculation.
Conclusions:
Using approved doses of Gd‐DOTA, the steady state RSST1‐signal in brain tissue is reached during the first pass and corresponds to the BVf. The first‐pass duration is sufficient to allow accurate BVf quantification. The RSST1‐method is appropriate for serial clinical studies since it allows fast and straightforward BVf quantification without arterial input function determination. This quantitative MRI method is particularly useful to assess the efficacy of antiangiogenic agents.
Nanoparticle (NP)-protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP's physicochemical features significantly impact on ...the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography-tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies.
Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or the internal segment of the globus pallidus (GPi) has been established as a highly effective symptomatic therapy for Parkinson's ...disease (PD). An intriguing biological aspect related to the DBS procedure is that a temporary contact establishes between surgical instruments and the surrounding brain tissue. In this exploratory study, we took advantage of this unique context to harvest brain material adhering to the stylet routinely used during surgery, and to examine the biological value of these samples, here referred to as "brain tissue imprints" (BTIs).
Nineteen BTIs from 12 STN- or GPi-electrode implanted patients were obtained in vivo during DBS surgery, without any modification of the surgical procedure. Immunofluorescence analyses confirmed that our approach allowed the harvesting of many neural cells including neurons harboring distinct neurotransmitter markers. Shotgun proteomic and transcriptomic analyses provided for the first time molecular information from DBS-associated brain samples, and confirmed the compatibility of this new type of sample with poly-omic approaches. The method appears to be safe and results consistent.
We here propose BTIs as original and highly valuable brain samples, and DBS-related brain imprinting as a new conceptual approach to biological research in living patients with PD.
Purpose:
In preclinical studies, the Rapid‐Steady‐State‐
T
1
(RSS
T
1
) MRI method has advantages over conventional MRI methods for blood volume fraction (BVf) mapping, since after contrast agent ...administration, the BVf is directly quantifiable from the signal amplitude corresponding to the vascular equilibrium magnetization. This study focuses on its clinical implementation and feasibility.
Methods:
Following sequence implementation on clinical Philips Achieva scanners, the RSS
T
1
‐method is assessed at 1.5 and 3 T in the follow‐up examination of neurooncological patients receiving 0.1–0.2 mmol/kg Gd‐DOTA to determine the threshold dose needed for cerebral BVf quantification. Confounding effects on BVf quantification such as transendothelial water exchange, transverse relaxation, and contrast agent extravasation are evaluated.
Results:
For a dose ≥0.13 mmol/kg at 1.5 T and ≥0.16 mmol/kg at 3 T, the RSS
T
1
‐signal time course in macrovessels and brain tissue with Gd‐DOTA impermeable vasculature reaches a steady state at maximum amplitude for about 8 s. In macrovessels, a BVf of 100% was obtained validating cerebral microvascular BVf quantification (3.5%–4.5% in gray matter and 1.5%–2.0% in white matter). In tumor tissue, a continuously increasing signal is detected, necessitating signal modeling for tumor BVf calculation.
Conclusions:
Using approved doses of Gd‐DOTA, the steady state RSS
T
1
‐signal in brain tissue is reached during the first pass and corresponds to the BVf. The first‐pass duration is sufficient to allow accurate BVf quantification. The RSS
T
1
‐method is appropriate for serial clinical studies since it allows fast and straightforward BVf quantification without arterial input function determination. This quantitative MRI method is particularly useful to assess the efficacy of antiangiogenic agents.
L'analyse protéomique du tissu cérébral pathologique dans la maladie de Parkinson (MP) est un enjeu majeur à l'identification des causes moléculaires de la dégénérescence en vue de développer des ...thérapies curatives. Jusqu'à présent, les études chez l'humain se sont limitées à l'analyse du cerveau post-mortem, trop souvent représentatif d'un stade très avancé de la maladie et potentiellement altéré par différents facteurs. Dans ce travail, nous avons exploité l'accès temporaire au cerveau parkinsonien durant l'implantation d'électrodes de stimulation, pour obtenir une information moléculaire du tissu cérébral, in vivo et à un stade moins avancé de la maladie. Afin d'optimiser notre stratégie, nous avons ensuite développé un outil dédié à la capture tissulaire dont l'efficacité et le caractère non lésionnel ont été validés in vivo chez le primate. Ce travail permet d'envisager l'analyse protéomique du cerveau parkinsonien « vivant » afin d'identifier les causes moléculaires de la MP. En revanche, cette approche tissulaire n'est pas envisageable pour un diagnostic en routine clinique. Aussi, de nombreux groupes s'intéressent à l'analyse protéomique du LCR en vue d'identifier des marqueurs diagnostiques. Dans cette optique, nous avons mis au point une stratégie, basée sur l'utilisation de nanoparticules (NPs) fonctionnalisées qui a permis un enrichissement considérable des profils protéiques observés en spectrométrie de masse. La reproductibilité et la possibilité d'automatiser intégralement la préparation des échantillons font de notre approche une solution adaptée à la recherche de marqueurs moléculaires diagnostiques de la MP dans le LCR. Nous avons aussi démontré l'intérêt de notre approche pour l'analyse protéomique du plasma et du globule rouge. Enfin, nous avons évalué la possibilité d'utiliser ces NPs in vivo, pour une capture des protéines directement dans la circulation sanguine.
Proteomics analysis of pathological brain tissue in Parkinson's disease (PD) is of great importance to understand the molecular aetiology of degeneration and to develop curative treatments. To date, published studies have been restricted to the analysis of human post-mortem tissue samples, frequently derived from advanced disease stage and potentially altered by several factors. In this project we took advantage of the temporary access to PD patient's brain during electrode implantation to obtain in vivo molecular information from cerebral tissue at earlier stage of the disease. We further developed a dedicated tool to improve our tissue harvesting approach, and validated its efficiency and non-lesion effects in vivo in monkeys. This work opens the way to the proteomic analysis of fresh human brain samples to elucidate molecular causes of degeneration in PD. However such tissue investigation approach remains invasive and cannot be used in routine clinical screening for PD diagnosis. Proteomics analysis of cerebrospinal fluid (CSF) constitutes a promising alternative to identify neuropathological diagnosis markers. For this purpose, we developed a nanoparticle-assisted strategy enabling the enrichment of CSF proteins detection by mass spectrometry. Reproducibility and high throughput potentiality of our approach demonstrate its compatibility with clinical proteomics for PD diagnosis biomarker research in CSF. We also demonstrated the interest of this NP strategy for plasma and red blood cells proteome analysis. Finally, we evaluated the ability to use these NPs for in vivo protein harvesting in blood.
Access to cerebral tissue is essential to better understand the molecular mechanisms associated with neurodegenerative diseases. In this study, we present, for the first time, a new tool designed to ...obtain molecular and cellular cerebral imprints in the striatum of anesthetized monkeys. The imprint is obtained during a spatially controlled interaction of a chemically modified micro-silicon chip with the brain tissue. Scanning electron and immunofluorescence microscopies showed homogeneous capture of cerebral tissue. Nano-liquid chromatography–tandem mass spectrometry (nano-LC-MS/MS) analysis of proteins harvested on the chip allowed the identification of 1158 different species of proteins. The gene expression profiles of mRNA extracted from the imprint tool showed great similarity to those obtained via the gold standard approach, which is based on post-mortem sections of the same nucleus. Functional analysis of the harvested molecules confirmed the spatially controlled capture of striatal proteins implicated in dopaminergic regulation. Finally, the behavioral monitoring and histological results establish the safety of obtaining repeated cerebral imprints in striatal regions. These results demonstrate the ability of our imprint tool to explore the molecular content of deep brain regions in vivo. They open the way to the molecular exploration of brain in animal models of neurological diseases and will provide complementary information to current data mainly restricted to post-mortem samples.
L'analyse protéomique du tissu cérébral pathologique dans la maladie de Parkinson (MP) est un enjeu majeur à l'identification des causes moléculaires de la dégénérescence en vue de développer des ...thérapies curatives. Jusqu'à présent, les études chez l'humain se sont limitées à l'analyse du cerveau post-mortem, trop souvent représentatif d'un stade très avancé de la maladie et potentiellement altéré par différents facteurs. Dans ce travail, nous avons exploité l'accès temporaire au cerveau parkinsonien durant l'implantation d'électrodes de stimulation, pour obtenir une information moléculaire du tissu cérébral, in vivo et à un stade moins avancé de la maladie. Afin d'optimiser notre stratégie, nous avons ensuite développé un outil dédié à la capture tissulaire dont l'efficacité et le caractère non lésionnel ont été validés in vivo chez le primate. Ce travail permet d'envisager l'analyse protéomique du cerveau parkinsonien " vivant " afin d'identifier les causes moléculaires de la MP. En revanche, cette approche tissulaire n'est pas envisageable pour un diagnostic en routine clinique. Aussi, de nombreux groupes s'intéressent à l'analyse protéomique du LCR en vue d'identifier des marqueurs diagnostiques. Dans cette optique, nous avons mis au point une stratégie, basée sur l'utilisation de nanoparticules (NPs) fonctionnalisées qui a permis un enrichissement considérable des profils protéiques observés en spectrométrie de masse. La reproductibilité et la possibilité d'automatiser intégralement la préparation des échantillons font de notre approche une solution adaptée à la recherche de marqueurs moléculaires diagnostiques de la MP dans le LCR. Nous avons aussi démontré l'intérêt de notre approche pour l'analyse protéomique du plasma et du globule rouge. Enfin, nous avons évalué la possibilité d'utiliser ces NPs in vivo, pour une capture des protéines directement dans la circulation sanguine.
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