Evaluation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of Glucosylceramide and Galactosylceramide Isoforms in Cerebrospinal Fluid of Parkinson's Disease Patients.

Mutations in GBA1, encoding glucocerebrosidase beta 1 (GCase), are the most common genetic risk factor for Parkinson's disease (PD). GCase dysfunction leads to an accumulation of glucosylceramide (GluCer) substrates in different organs and fluids. Despite the challenges in quantifying GluCer isoforms in biological samples, their potential clinical interest as PD biomarkers justifies the development of robust assays. An extensively evaluated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying 14 GluCer and galactosylceramide (GalCer) isoforms in human cerebrospinal fluid (CSF) samples is presented. Sample pretreatment, HPLC, and MS/MS parameters were optimized. Evaluation was performed according to the recommendations of the Clinical and Laboratory Standards Institute and European Medicines Agency guidelines. Four 7-point calibration curves were generated, with a linearity interval from 2.5 to 200 nM (R2 ≥ 0.995). The limit of quantification was set at 5 nM. Between-run precision and accuracy were up to 12.5 and 9%, respectively. After method validation, we measured the levels of GluCer and GalCer isoforms in CSF human samples, including 6 healthy controls (HC), 22 idiopathic GBA1 wild-type PD (iPD) patients, and 5 GBA1-associated PD (PD-GBA) patients. GluCer/GalCer median ratios were found to be higher in the CSF of PD-GBA patients, particularly in severe GBA1 mutations, than those in iPD and HC. The observed trends in GluCer/GalCer ratios among groups provide novel information for the comprehensive analysis of sphingolipids as potential biomarkers of PD.

Phenotypic effect of GBA1 variants in individuals with and without Parkinson's disease: The RAPSODI study.

BACKGROUND: Variants in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD). They are also risk factors for Parkinson's disease (PD), and modify the expression of the PD phenotype. The penetrance of GBA1 variants in PD is incomplete, and the ability to determine who among GBA1 variant carriers are at higher risk of developing PD, would represent an advantage for prognostic and trial design purposes. OBJECTIVES: To compare the motor and non-motor phenotype of GBA1 carriers and non-carriers. METHODS: We present the cross-sectional results of the baseline assessment from the RAPSODI study, an online assessment tool for PD patients and GBA1 variant carriers. The assessment includes clinically validated questionnaires, a tap-test, the University of Pennsyllvania Smell Identification Test and cognitive tests. Additional, homogeneous data from the PREDICT-PD cohort were included. RESULTS: A total of 379 participants completed all parts of the RAPSODI assessment (89 GBA1-negative controls, 169 GBA1-negative PD, 47 GBA1-positive PD, 47 non-affected GBA1 carriers, 27 GD). Eighty-six participants were recruited through PREDICT-PD (43 non-affected GBA1 carriers and 43 GBA1-negative controls). GBA1-positive PD patients showed worse performance in visual cognitive tasks and olfaction compared to GBA1-negative PD patients. No differences were detected between non-affected GBA1 carriers carriers and GBA1-negative controls. No phenotypic differences were observed between any of the non-PD groups. CONCLUSIONS: Our results support previous evidence that GBA1-positive PD has a specific phenotype with more severe non-motor symptoms. However, we did not reproduce previous findings of more frequent prodromal PD signs in non-affected GBA1 carriers.

Influence of initial clinical suspicion on the diagnostic yield of laboratory enzymatic testing in lysosomal storage disorders. Experience from a multispecialty hospital.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD.

PHD3-SUMO conjugation represses HIF1 transcriptional activity independently of PHD3 catalytic activity.

By controlling HIFα hydroxylation and stability, the prolyl hydroxylase domain (PHD)-containing proteins are essential to the maintenance of oxygen homeostasis; therefore these enzymes are tightly regulated. Small ubiquitin-like modifier (SUMO) is a 10-kDa protein readily conjugated to lysine residues of the targeted proteins in a process termed SUMOylation. In this study, we introduce SUMO conjugation as a novel regulator of PHD3 (also known as EGLN3). PHD3 SUMOylation occurs at a cluster of four lysines at the C-terminal end of the protein. Furthermore, PHD3 SUMOylation by SUMO2 or SUMO3 contributes to PHD3-mediated repression of HIF1-dependent transcriptional activity. Interestingly, PHD3-SUMO conjugation does not affect PHD3 hydroxylase activity or HIF1α stability, providing new evidence for a dual role of PHD3 in HIF1 regulation. Moreover, we show that hypoxia modulates PHD3-SUMO conjugation and that this modification inversely correlates with HIF1 activation. PHD3 SUMOylation highlights a new and additional layer of regulation that is likely required to fine-tune HIF function.

Sex-Specific Microglial Responses to Glucocerebrosidase Inhibition: Relevance to GBA1-Linked Parkinson's Disease.

Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.

Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Biomarkers in Fabry Disease. Implications for Clinical Diagnosis and Follow-up.

Fabry disease (FD) is a lysosomal storage disorder caused by deficient alpha-galactosidase A activity in the lysosome due to mutations in the GLA gene, resulting in gradual accumulation of globotriaosylceramide and other derivatives in different tissues. Substrate accumulation promotes different pathogenic mechanisms in which several mediators could be implicated, inducing multiorgan lesions, mainly in the kidney, heart and nervous system, resulting in clinical manifestations of the disease. Enzyme replacement therapy was shown to delay disease progression, mainly if initiated early. However, a diagnosis in the early stages represents a clinical challenge, especially in patients with a non-classic phenotype, which prompts the search for biomarkers that help detect and predict the evolution of the disease. We have reviewed the mediators involved in different pathogenic mechanisms that were studied as potential biomarkers and can be easily incorporated into clinical practice. Some accumulation biomarkers seem to be useful to detect non-classic forms of the disease and could even improve diagnosis of female patients. The combination of such biomarkers with some response biomarkers, may be useful for early detection of organ injury. The incorporation of some biomarkers into clinical practice may increase the capacity of detection compared to that currently obtained with the established diagnostic markers and provide more information on the progression and prognosis of the disease.