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.

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.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons.

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Triplication of DYRK1A causes retinal structural and functional alterations in Down syndrome.

Down syndrome (DS) results from the triplication of approximately 300 human chromosome 21 (Hsa21) genes and affects almost all body organs. Children with DS have defects in visual processing that may have a negative impact on their daily life and cognitive development. However, there is little known about the genes and pathogenesis underlying these defects. Here, we show morphometric in vivo data indicating that the neural retina is thicker in DS individuals than in the normal population. A similar thickening specifically affecting the inner part of the retina was also observed in a trisomic model of DS, the Ts65Dn mouse. Increased retinal size and cellularity in this model correlated with abnormal retinal function and resulted from an impaired caspase-9-mediated apoptosis during development. Moreover, we show that mice bearing only one additional copy of Dyrk1a have the same retinal phenotype as Ts65Dn mice and normalization of Dyrk1a gene copy number in Ts65Dn mice completely rescues both, morphological and functional phenotypes. Thus, triplication of Dyrk1a is necessary and sufficient to cause the retinal phenotype described in the trisomic model. Our data demonstrate for the first time the implication of DYRK1A overexpression in a developmental alteration of the central nervous system associated with DS, thereby providing insights into the aetiology of neurosensorial dysfunction in a complex disease.

Investigating the impact of physical activity on mitochondrial function in Parkinson's disease (PARKEX): Study protocol for A randomized controlled clinical trial.

Parkinson's disease (PD) is characterized by the progressive dopaminergic neuron degeneration, resulting in striatal dopamine deficiency. Mitochondrial dysfunction and oxidative stress are associated with PD pathogenesis. Physical activity (PA) has been shown to ameliorate neurological impairments and to impede age-related neuronal loss. In addition, skin fibroblasts have been identified as surrogate indicators of pathogenic processes correlating with clinical measures. The PARKEX study aims to compare the effects of two different PA programs, analyzing the impact on mitochondrial function in patients' skin fibroblasts as biomarkers for disease status and metabolic improvement. Early-stage PD patients (n = 24, H&Y stage I to III) will be randomized into three age- and sex-matched groups. Group 1 (n = 8) will undergo basic physical training (BPT) emphasizing strength and resistance. Group 2 (n = 8) will undergo BPT combined with functional exercises (BPTFE), targeting the sensorimotor pathways that are most affected in PD (proprioception-balance-coordination) together with cognitive and motor training (Dual task training). Group 3 (n = 8) will serve as control (sedentary group; Sed). Participants will perform three sessions per week for 12 weeks. Assessment of motor function, quality of life, sleep quality, cognitive aspects and humor will be conducted pre- and post-intervention. Patient skin fibroblasts will be collected before and after the intervention and characterized in terms of metabolic remodeling and mitochondrial bioenergetics. Ethical approval has been given to commence this study. This trial is registered at clinicaltrials.gov (NCT05963425). Trial registration. https://classic.clinicaltrials.gov/ct2/history/NCT05963425.

Sex Differences in Motor and Non-Motor Symptoms among Spanish Patients with Parkinson's Disease.

BACKGROUND AND OBJECTIVE: Sex plays a role in Parkinson's disease (PD) mechanisms. We analyzed sex difference manifestations among Spanish patients with PD. PATIENTS AND METHODS: PD patients who were recruited from the Spanish cohort COPPADIS from January 2016 to November 2017 were included. A cross-sectional and a two-year follow-up analysis were conducted. Univariate analyses and general linear model repeated measure were used. RESULTS: At baseline, data from 681 PD patients (mean age 62.54 ± 8.93) fit the criteria for analysis. Of them, 410 (60.2%) were males and 271 (39.8%) females. There were no differences between the groups in mean age (62.36 ± 8.73 vs. 62.8 ± 9.24; p = 0.297) or in the time from symptoms onset (5.66 ± 4.65 vs. 5.21 ± 4.11; p = 0.259). Symptoms such as depression (p < 0.0001), fatigue (p < 0.0001), and pain (p < 0.00001) were more frequent and/or severe in females, whereas other symptoms such as hypomimia (p < 0.0001), speech problems (p < 0.0001), rigidity (p < 0.0001), and hypersexuality (p < 0.0001) were more noted in males. Women received a lower levodopa equivalent daily dose (p = 0.002). Perception of quality of life was generally worse in females (PDQ-39, p = 0.002; EUROHIS-QOL8, p = 0.009). After the two-year follow-up, the NMS burden (Non-Motor Symptoms Scale total score) increased more significantly in males (p = 0.012) but the functional capacity (Schwab and England Activities of Daily Living Scale) was more impaired in females (p = 0.001). CONCLUSION: The present study demonstrates that there are important sex differences in PD. Long-term prospective comparative studies are needed.

DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles.

Dual-specificity tyrosine-regulated kinases (DYRKs) comprise a family of protein kinases within the CMGC group of the eukaryotic kinome. Members of the DYRK family are found in 4 (animalia, plantae, fungi, and protista) of the 5 main taxa or kingdoms, and all DYRK proteins studied to date share common structural, biochemical, and functional properties with their ancestors in yeast. Recent work on DYRK proteins indicates that they participate in several signaling pathways critical for developmental processes and cell homeostasis. In this review, we focus on the DYRK family of proteins from an evolutionary, biochemical, and functional point of view and discuss the most recent, relevant, and controversial contributions to the study of these kinases.

New Avenues for Parkinson's Disease Therapeutics: Disease-Modifying Strategies Based on the Gut Microbiota.

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder that currently affects 1% of the population over the age of 60 years, and for which no disease-modifying treatments exist. Neurodegeneration and neuropathology in different brain areas are manifested as both motor and non-motor symptoms in patients. Recent interest in the gut-brain axis has led to increasing research into the gut microbiota changes in PD patients and their impact on disease pathophysiology. As evidence is piling up on the effects of gut microbiota in disease development and progression, another front of action has opened up in relation to the potential usage of microbiota-based therapeutic strategies in treating gastrointestinal alterations and possibly also motor symptoms in PD. This review provides status on the different strategies that are in the front line (i.e., antibiotics; probiotics; prebiotics; synbiotics; dietary interventions; fecal microbiota transplantation, live biotherapeutic products), and discusses the opportunities and challenges the field of microbiome research in PD is facing.

Serum metabolic biomarkers for synucleinopathy conversion in isolated REM sleep behavior disorder.

Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is a prodromal stage of Lewy-type synucleinopathies (LTS), which can present either with an initial predominant parkinsonism (Parkinson's disease (PD)) or dementia (dementia with Lewy bodies (DLB)). To provide insights into the underlying pathogenic mechanisms, the lipoprotein and protein glycosylation profile of 82 iRBD patients, collected before and/or after their conversion to an overt LTS, and 29 matched control serum samples were assessed by nuclear magnetic resonance (NMR) spectroscopy. Data were statistically analyzed to identify altered metabolites and construct predictive models. Univariant analysis detected no differences between iRBD patients with an LTS compared to controls. However, significant differences were found when the analysis distinguished between iRBD patients that manifested initially predominant parkinsonism (pre-PD) or dementia (pre-DLB). Significant differences were also found in the analysis of paired iRBD samples pre- and post-LTS diagnosis. Predictive models were built and distinguished between controls and pre-DLB patients, and between pre-DLB and pre-PD patients. This allowed a prediction of the possible future clinical outcome of iRBD patients. We provide evidence of altered lipoprotein and glycosylation profiles in subgroups of iRBD patients. Our results indicate that metabolic alterations and inflammation are involved in iRBD pathophysiology, and suggest biological differences underlying the progression of LTS in iRBD patients. Our data also indicate that profiling of serum samples by NMR may be a useful tool for identifying short-term high-risk iRBD patients for conversion to parkinsonism or dementia.

Validation of a Reversed Phase UPLC-MS/MS Method to Determine Dopamine Metabolites and Oxidation Intermediates in Neuronal Differentiated SH-SY5Y Cells and Brain Tissue.

Dopamine is a key neurotransmitter in the pathophysiology of various neurological disorders such as addiction or Parkinson's disease. Disturbances in its metabolism could lead to dopamine accumulation in the cytoplasm and an increased production of o-quinones and their derivatives, which have neurotoxic potential and act as precursors in neuromelanin synthesis. Thus, quantification of the dopaminergic metabolism is essential for monitoring changes that may contribute to disease development. Here, we developed and validated an UPLC-MS/MS method to detect and quantify a panel of eight dopaminergic metabolites, including the oxidation product aminochrome. Our method was validated in differentiated SH-SY5Y cells and mouse brain tissue and was then employed in brain samples from humans and rats to ensure method reliability in different matrices. Finally, to prove the biological relevance of our method, we determined metabolic changes in an in vitro cellular model of dopamine oxidation/neuromelanin production and in human postmortem samples from Parkinson's disease patients. The current study provides a validated method to simultaneously monitor possible alterations in dopamine degradation and o-quinone production pathways that can be applied to in vitro and in vivo experimental models of neurological disorders and human brain samples.

Intracellular crowding by age-dependent neuromelanin accumulation disrupts neuronal proteostasis and triggers Parkinson disease pathology.

In Parkinson disease (PD), there is a preferential degeneration of neurons that contain the dark-brown cytoplasmic pigment neuromelanin, in particular dopaminergic neurons of the substantia nigra (SN), the loss of which leads to the typical motor symptoms of the disease and constitutes the cardinal pathological diagnostic criterion for PD. Neuromelanin is generally considered a byproduct of dopamine oxidative metabolism and, in humans, it is first detected in early childhood and accumulates progressively with age until occupying most of the neuronal cytoplasm, as neurons apparently lack the means to degrade or eliminate this pigment. Aging is the main risk factor for developing PD, but the molecular substrate underlying this link remains unknown. Despite the close and long-established association between neuromelanin and PD, the potential contribution of neuromelanin to PD pathogenesis has remained elusive because, in contrast to humans, common laboratory animal species, such as rodents, lack neuromelanin. To overcome this major limitation, we have recently generated the first experimental in vivo rodent model exhibiting age-dependent production and accumulation of human-like neuromelanin within PD-vulnerable dopaminergic nigral neurons, at levels up to those reached in elderly humans.

Autophagic- and Lysosomal-Related Biomarkers for Parkinson's Disease: Lights and Shadows.

Parkinson's disease (PD) is a neurodegenerative disorder that currently affects 1% of the population over the age of 60 years, for which no disease-modifying treatments exist. This lack of effective treatments is related to the advanced stage of neurodegeneration existing at the time of diagnosis. Thus, the identification of early stage biomarkers is crucial. Biomarker discovery is often guided by the underlying molecular mechanisms leading to the pathology. One of the central pathways deregulated during PD, supported both by genetic and functional studies, is the autophagy-lysosomal pathway. Hence, this review presents different studies on the expression and activity of autophagic and lysosomal proteins, and their functional consequences, performed in peripheral human biospecimens. Although most biomarkers are inconsistent between studies, some of them, namely HSC70 levels in sporadic PD patients, and cathepsin D levels and glucocerebrosidase activity in PD patients carrying GBA mutations, seem to be consistent. Hence, evidence exists that the impairment of the autophagy-lysosomal pathway underlying PD pathophysiology can be detected in peripheral biosamples and further tested as potential biomarkers. However, longitudinal, stratified, and standardized analyses are needed to confirm their clinical validity and utility.

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

In 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.

The E3 Ubiquitin Ligases TRIM17 and TRIM41 Modulate α-Synuclein Expression by Regulating ZSCAN21.

Although accumulating data indicate that increased α-synuclein expression is crucial for Parkinson disease (PD), mechanisms regulating the transcription of its gene, SNCA, are largely unknown. Here, we describe a pathway regulating α-synuclein expression. Our data show that ZSCAN21 stimulates SNCA transcription in neuronal cells and that TRIM41 is an E3 ubiquitin ligase for ZSCAN21. In contrast, TRIM17 decreases the TRIM41-mediated degradation of ZSCAN21. Silencing of ZSCAN21 and TRIM17 consistently reduces SNCA expression, whereas TRIM41 knockdown increases it. The mRNA levels of TRIM17, ZSCAN21, and SNCA are simultaneously increased in the midbrains of mice following MPTP treatment. In addition, rare genetic variants in ZSCAN21, TRIM17, and TRIM41 genes occur in patients with familial forms of PD. Expression of variants in ZSCAN21 and TRIM41 genes results in the stabilization of the ZSCAN21 protein. Our data thus suggest that deregulation of the TRIM17/TRIM41/ZSCAN21 pathway may be involved in the pathogenesis of PD.

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease.

The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson's disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson's disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson's disease pathogenesis.

Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons.

Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.

α-Synuclein-induced down-regulation of Nurr1 disrupts GDNF signaling in nigral dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) and its close relative neurturin are currently in clinical trials for neuroprotection in patients with Parkinson disease (PD). However, in animal models of PD, GDNF fails to protect nigral dopamine (DA) neurons against α-synuclein-induced neurodegeneration. Using viral vector delivery of human wild-type α-synuclein to nigral DA neurons in rats, we show that the intracellular response to GDNF is blocked in DA neurons that overexpress α-synuclein. This block is accompanied by reduced expression of the transcription factor Nurr1 and its downstream target, the GDNF receptor Ret. We found that Ret expression was also reduced in nigral DA neurons in PD patients. Conditional knockout of Nurr1 in mice resulted in reduced Ret expression and blockade of the response to GDNF, whereas overexpression of Nurr1 restored signaling, providing protection of nigral DA neurons against α-synuclein toxicity. These results suggest that Nurr1 is a regulator of neurotrophic factor signaling and a key player in the cellular defense against α-synuclein toxicity.

Regulated segregation of kinase Dyrk1A during asymmetric neural stem cell division is critical for EGFR-mediated biased signaling.

Stem cell division can result in two sibling cells exhibiting differential mitogenic and self-renewing potential. Here, we present evidence that the dual-specificity kinase Dyrk1A is part of a molecular pathway involved in the regulation of biased epidermal growth factor receptor (EGFR) signaling in the progeny of dividing neural stem cells (NSC) of the adult subependymal zone (SEZ). We show that EGFR asymmetry requires regulated sorting and that a normal Dyrk1a dosage is required to sustain EGFR in the two daughters of a symmetrically dividing progenitor. Dyrk1A is symmetrically or asymmetrically distributed during mitosis, and biochemical analyses indicate that it prevents endocytosis-mediated degradation of EGFR by a mechanism that requires phosphorylation of the EGFR signaling modulator Sprouty2. Finally, Dyrk1a heterozygous NSCs exhibit defects in self-renewal, EGF-dependent cell-fate decisions, and long-term persistence in vivo, suggesting that symmetrical divisions play a role in the maintenance of the SEZ reservoir.

Sprouty2-mediated inhibition of fibroblast growth factor signaling is modulated by the protein kinase DYRK1A.

Raf-MEK-extracellular signal-regulated kinase (Erk) signaling initiated by growth factor-engaged receptor tyrosine kinases (RTKs) is modulated by an intricate network of positive and negative feedback loops which determine the specificity and spatiotemporal characteristics of the intracellular signal. Well-known antagonists of RTK signaling are the Sprouty proteins. The activity of Sprouty proteins is modulated by phosphorylation. However, little is known about the kinases responsible for these posttranslational modifications. We identify DYRK1A as one of the protein kinases of Sprouty2. We show that DYRK1A interacts with and regulates the phosphorylation status of Sprouty2. Moreover, we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation site in vitro and in vivo. This site is functional, since its mutation enhanced the repressive function of Sprouty2 on fibroblast growth factor (FGF)-induced Erk signaling. Further supporting the idea of a functional interaction, DYRK1A and Sprouty2 are present in protein complexes in mouse brain, where their expression overlaps in several structures. Moreover, both proteins copurify with the synaptic plasma membrane fraction of a crude synaptosomal preparation and colocalize in growth cones, pointing to a role in nerve terminals. Our results suggest, therefore, that DYRK1A positively regulates FGF-mitogen-activated protein kinase signaling by phosphorylation-dependent impairment of the inhibitory activity of Sprouty2.

The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development.

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome.