Search Results (24)

Gaucher disease is a prototypical lysosomal sphingolipid storage disorder caused by pathogenic variants in GBA1, resulting in glucocerebrosidase deficiency and accumulation of bioactive lipids, including glucosylceramide and glucosylsphingosine (lyso-Gb1). While non-neuronopathic Gaucher disease is effectively managed with enzyme replacement and substrate reduction therapies, neuronopathic forms remain largely refractory to treatment due to progressive central nervous system (CNS) involvement and limited penetration of current therapies across the blood–brain barrier. Disease pathobiology extends beyond lysosomal substrate accumulation to encompass dysregulated sphingolipid signaling, particularly sphingosine-1-phosphate (S1P)-mediated “inside-out” signaling, alongside neuroinflammation, oxidative stress, and glial activation, which collectively drive neurodegeneration. In this review, we synthesize current knowledge on sphingolipid metabolism and signaling in neuronopathic Gaucher disease and integrate these mechanisms into a three-tier, CNS-focused biomarker framework. The first tier comprises substrate-proximal markers of lysosomal burden (lyso-Gb1), which reflect GCase deficiency and correlate with systemic disease severity but incompletely capture CNS pathology. The second tier comprises markers of glial activation and neuroinflammation (glial fibrillary acidic protein [GFAP], glycoprotein non-metastatic melanoma protein B [GPNMB]), which reflect the downstream neuroimmune response to sphingolipid accumulation. The third tier comprises markers of neuroaxonal injury (neurofilament light chain [NfL]), which index irreversible neuronal damage as the terminal consequence of uncontrolled CNS disease. Together, these tiers map distinct but mechanistically interconnected stages of disease progression, from lysosomal dysfunction through glial activation to neuroaxonal loss, enabling stage-specific interpretation of biomarker signals that single-analyte approaches cannot provide. We further examine how S1P-mediated inside-out signaling links intracellular lipid dysregulation to extracellular neuroimmune and neurovascular responses and how the blood–brain barrier shapes compartment-dependent biomarker behavior across cerebrospinal fluid and blood. By grounding biomarker selection in this mechanistic cascade, the framework provides explicit criteria for pairing analytes across tiers, interpreting discordance between peripheral and CNS compartments, and designing multi-modal endpoints for clinical trials of CNS-penetrant therapies. Despite these advances, significant challenges remain, including limited longitudinal datasets, variability in assay methodologies, and incomplete validation of biomarkers as surrogates of CNS disease progression. Addressing these gaps will require harmonized, multi-modal approaches integrating biochemical, functional, and imaging measures. By positioning neuronopathic Gaucher disease as a model of sphingolipid-driven neurodegeneration, this review highlights opportunities for biomarker-guided therapeutic development relevant to Gaucher disease and the broader spectrum of sphingolipid-associated neurological disorders. Full article

A highly selective and sensitive compact immunosensing strategy was developed for the determination of DJ-1, a potential biomarker of Parkinson’s disease, one of the leading neurodegenerative disorders, using a portable potentiostat. Initially, screen-printed carbon electrodes (SPCEs) were modified with gold nanoparticles (AuNPs), followed by functionalization with 4-mercapto-1-butanol (MOH). Subsequently, the AuNPs-doped and hydroxyl-functionalized electrodes were treated with 3-glycidoxypropyltrimethoxysilane (GPTMS) to facilitate immobilization of anti-DJ-1 antibodies. Immobilization steps were monitored using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) performed on a bench potentiostat, while the entire analytical performance of the developed biosensor system and its response in artificial cerebrospinal fluid (aCSF) were evaluated by monitoring cathodic current changes with a portable electrochemical reader. The resulting biorecognition element enabled the detection of DJ-1 within the concentration range of 0.001 to 0.3 ng/mL, based on cathodic current changes, achieving a limit of detection as low as 0.00059 ng/mL. Surface morphology and elemental composition alterations were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and energy-dispersive X-ray spectroscopy (EDX). A notable advantage of this GPTMS@AuNPs-based biosensor system is its prolonged storage stability and its capability to accurately quantify DJ-1 in artificial cerebrospinal fluid samples, with recovery rates ranging from 98.66% to 123.3%. Full article

The flow behavior of fluids can be characterized by rheology and is especially used in the field of polymeric materials. This study focused on characterizing cerebrospinal fluid (CSF) of patients who developed hydrocephalus after subarachnoid hemorrhage (SAH) with rheology. Samples were drawn from an external ventricular drainage (EVD) at four pre-defined time points after the initial hemorrhage. The CSF samples were analyzed using a rotational rheometer with a double gap geometry. In addition to the characterization of viscoelastic parameters, the cumulative storage factor was calculated to determine the interactions in the fluid. In order to investigate the temperature dependence of the CSF properties, the oscillatory measurements were implemented at certain temperatures that simulated specific conditions, such as 5 °C, at which temperature the CSF samples were stored; 35 °C for hypothermic conditions; 37 °C for physiologic conditions; and 40 °C for elevated body temperature. The overall goal was to evaluate whether rheology-based parameters may help in the prediction of shunt dependence for post-hemorrhagic hydrocephalus patients. For this aim, rheological parameters were correlated to certain laboratory parameters, such as erythrocyte and leukocyte count, glucose, lactate, and total protein concentration. Full article

Background/Objectives: Longitudinal metabolomics analysis offers valuable insights into how metabolic pathways change according to age and health status. However, metabolite levels can fluctuate due to biological factors (e.g., age, diet, and health status) and technical factors (e.g., sample handling, storage times, and instrument performance), with some metabolites exhibiting greater sensitivity to these sources of variability than others. This study aimed to characterize the longitudinal and technical stability of untargeted plasma and cerebrospinal fluid (CSF) metabolites and to identify a subset that remains reliable over the extended time scales required for epidemiological research. Methods: Untargeted ultrahigh-performance liquid chromatography–mass spectrometry (LC-MS) metabolomic profiles were available from multiple visits in the Wisconsin Registry for Alzheimer’s Prevention (WRAP) and Wisconsin Alzheimer’s Disease Research Center (ADRC) studies. For this analysis, we constructed a subset of generally healthy participants with samples drawn at four time points (~2.5 years apart): two visits analyzed in 2017 and two visits analyzed in 2023, corresponding to two distinct analytical waves. We computed Rothery’s intraclass correlation coefficients (ICCs) to quantify intra-wave and inter-wave stability, evaluated pooled quality-control (QC) variation, classified metabolite stability by established thresholds, and developed a composite score integrating longitudinal stability and susceptibility to technical variance. Results: Across all metabolites, median stability was classified as ‘fair’ (Rothery’s ρ > 0.40 to ≤0.75) for both plasma and CSF. Although analytical batches were bridged using pooled QC samples, inter-wave stability was significantly lower than intra-wave stability, reflecting increased technical variability across waves. Using the composite score, we identified subsets of metabolites with ‘excellent’ stability and low susceptibility to batch effects in plasma and CSF. Stability patterns varied across biochemical super pathways. Conclusions: This work highlights metabolites suitable for long-term epidemiological studies and informs experimental design and analytical strategies for combining data across cohorts and analytical batches. Full article

Background/Objectives: Nanocrystals (NCs) are a relatively underexplored yet adaptable platform with broad potential for various applications. Currently, the surface modification of NCs leads to the development of versatile platforms capable of enhancing targeted delivery potential and supporting the advancement of precision medicine. With this in mind, this study focused on the design and surface functionalization of a resveratrol (RSV) NC selected for its antioxidant and neuroprotective effects. Methods: The design of the RSV NC was assessed by the Quality by Design approach. With the aim of intranasal administration, we assessed the RSV NC functionalization with a cationic poly (amino acid) belonging to the class of cell-penetrating peptides. Both naked and surface-modified RSV nanosuspensions were characterized in terms of mucoadhesion, behavior in artificial cerebrospinal fluid, crystallinity, solubility, and storage stability. The scavenging activity (%) of neat RSV and its nanosized forms was measured using the DPPH assay. Results: RSV NCs were successfully designed, producing truncated cubic crystals (~240 nm) with an ~80% drug content. Functionalization was efficiently achieved with poly-l-arginine hydrochloride as revealed by DSC and FTIR and resulted in a positively charged nanosuspension. Nanonization technology improved drug solubility in water and did not affect RSV scavenging activity. Technological characterization demonstrated that both nanosuspensions present suitable properties for intranasal administration in terms of particle size, mucoadhesive tendency, and stability in artificial cerebrospinal fluid. An MTT assay revealed the safety of all treatments in human microglia (HMC3) cells. Conclusions: RSV NCs’ functionalization enhanced their brain delivery potential, establishing a promising platform to improve therapeutic outcomes in neurodegenerative diseases. Full article

Due to their clinical heterogeneity, nonspecific symptoms, and the limitations of existing biomarkers and imaging modalities, metabolic brain diseases (MBDs), such as mitochondrial encephalopathies, lysosomal storage disorders, and glucose metabolism syndromes, pose significant diagnostic challenges. This review examines the growing potential of cell-free DNA (cfDNA) derived from cerebrospinal fluid (CSF) epigenetic profiling as a dynamic, cell-type-specific, minimally invasive biomarker approach for MBD diagnosis and monitoring. We review important technological platforms and their use in identifying CNS-specific DNA methylation patterns indicative of neuronal injury, neuroinflammation, and metabolic reprogramming, including cfMeDIP-seq, enzymatic methyl sequencing (EM-seq), and targeted bisulfite sequencing. By synthesizing current findings across disorders such as MELAS, Niemann–Pick disease, Gaucher disease, GLUT1 deficiency syndrome, and diabetes-associated cognitive decline, we highlight the superior diagnostic and prognostic resolution offered by CSF cfDNA methylation signatures relative to conventional CSF markers or neuroimaging. We also address technical limitations, interpretive challenges, and translational barriers to clinical implementation. Ultimately, this review explores CSF cfDNA epigenetic analysis as a liquid biopsy modality. The central objective is to assess whether epigenetic profiling of CSF-derived cfDNA can serve as a reliable and clinically actionable biomarker for improving the diagnosis and longitudinal monitoring of metabolic brain diseases. Full article

Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson’s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood–brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD. Full article

Background/Objectives: Fabry disease (FD) is a lysosomal storage disorder often associated with early-onset neuropathic pain, attributed to small fibre neuropathy (SFN). The dorsal root ganglion (DRG) has emerged as a critical site of early pathophysiological involvement in FD, with structural and functional alterations implicated in the development of neuropathic symptoms. This exploratory study introduces DRG proton density (DRG-PD) as a novel MRI-derived biomarker and evaluates its association with SFN. Methods: Eighty genetically confirmed FD patients underwent high-resolution 3T MRI with DRG-PD quantification at the lumbosacral levels L5 and S1. DRG-PD was derived from B1-corrected multi-echo spin echo sequences and normalised to cerebrospinal fluid intensity. All patients underwent clinical, biochemical and histological evaluation to determine SFN status. Associations between DRG imaging parameters and clinical variables were analysed using correlation and regression models. Diagnostic performance was evaluated using receiver operating characteristic curve analysis. Results: DRG-PD values were significantly increased in patients with classical FD and SFN, demonstrating a large effect size (Cliff’s δ = 0.92) and excellent discriminatory performance (AUC = 0.96). In contrast, DRG volume and T2 relaxation time were not significantly associated with SFN status. DRG-PD remained an independent predictor of SFN in multivariable logistic regression (p = 0.019). Conclusions: DRG-PD is a non-invasive correlate of SFN in classical FD. It may provide superior diagnostic value compared to existing MRI metrics and reflects proximal ganglionic pathology not captured by distal histological assessments. Full article

Alzheimer’s disease (AD) is a progressive irreversible neurodegenerative disorder that represents a major global public health concern. Traditionally, AD is diagnosed using cerebrospinal fluid biomarker analysis or brain imaging modalities. Recently, less burdensome, more widely available blood biomarker (BBM) assays for amyloid-beta (Aβ42/40) and phosphorylated-tau concentrations have been found to accurately identify the presence/absence of brain amyloid plaques and tau tangles and have helped to streamline AD diagnosis. However, few BBMs have been rigorously analytically validated. Herein, we report the analytical validation of a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) multiplex method for quantifying plasma phosphorylated-tau217 (p-tau217) and non-phosphorylated-tau217 (np-tau217) peptide concentrations. We combined the p-tau217/np-tau217 concentrations ratio (%p-tau217) and the previously validated LC-MS/MS multiplex assay for plasma Aβ42/40 into a new multianalyte assay with algorithmic analysis (MAAA; PrecivityAD2™ test) that identifies brain amyloid status based on brain amyloid positron emission tomography. We found (a) the %p-tau217 assay is precise, accurate, sensitive, and linear over a wide analytical measurement range, and free from carryover and interference; (b) the pre-analytical specimen collection, processing, storage, and shipping conditions that maintain plasma tau peptide stability; and (c) using the measured analytical imprecision for plasma Aβ42/40 and p-tau217/np-tau217 levels in a worst-case scenario model, the PrecivityAD2 test algorithm for amyloid pathology classification changed for only 3.5% of participants from brain amyloid positive to negative, or from negative to positive. The plasma sample preparation and LC-MS/MS methods underlying the PrecivityAD2 test are suitable for use in the clinical laboratory and valid for the test’s intended purpose: to aid in the diagnostic evaluation of individuals aged 55 and older with signs or symptoms of mild cognitive impairment or dementia. Full article

Background: Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world’s fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. We aimed to measure T-tau protein using CLEA and to elucidate its merits and limitations. Methods: We randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of cerebrospinal fluid specimens. Additionally, we used Western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze–thaw effects in three selected samples. Results: In 172 patients, 172 samples were extracted, with each patient providing only one sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring T-tau protein levels. In addition, ELISA and CLEA are similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2000 pg/mL, leading to low reproducibility during dilution analysis. Conclusions: Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze–thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials. Full article

Cerebrospinal fluid plays a crucial role in protecting the central nervous system (CNS) by providing mechanical support, acting as a shock absorber, and transporting nutrients and waste products. It is produced in the ventricles of the brain and circulates through the brain and spinal cord in a continuous flow. In the current review, we presented basic concepts related to cerebrospinal fluid history, cerebrospinal fluid production, circulation, and its main components, the role of the blood–brain barrier and the blood–cerebrospinal fluid barrier in the maintenance of cerebrospinal fluid homeostasis, and the utility of Albumin Quotient (Q_Alb) evaluation in the diagnosis of CNS diseases. We also discussed the collection of cerebrospinal fluid (type, number of tubes, and volume), time of transport to the laboratory, and storage conditions. Finally, we briefly presented the role of cerebrospinal fluid examination in CNS disease diagnosis of various etiologies and highlighted that research on identifying cerebrospinal fluid biomarkers indicating disease presence or severity, evaluating treatment effectiveness, and enabling understanding of pathogenesis and disease mechanisms is of great importance. Thus, in our opinion, research on cerebrospinal fluid is still necessary for both the improvement of CNS disease management and the discovery of new treatment options. Full article

Cerebrospinal fluid-based real-time quaking-induced conversion (CSF RT-QuIC) is currently the most prominent method for early detection of sporadic Creutzfeldt–Jakob disease (sCJD), the most common prion disease. CSF RT-QuIC delivers high sensitivity (>90%) and specificity (100%), which has been demonstrated by large ring-trial studies testing probable and definitive sCJD cohorts. Following the inclusion of CSF RT-QuIC in the revised European CJD Surveillance Network diagnostic criteria for sCJD, it has become a standard diagnostic procedure in many prion disease reference or surveillance centers around the world. In this study, we present the implementation of the second-generation CSF RT-QuIC (commonly known as Improved QuIC or IQ) at the Danish Reference Center for Prion Diseases (DRCPD). The method’s sensitivity and specificity were evaluated and validated by analyzing 63 CSF samples. These 63 samples were also analyzed at the National CJD Research and Surveillance Unit (NCJDRSU), based at the University of Edinburgh, UK; analysis was carried out using the first generation or previous CSF RT-QuIC method (PQ). The sensitivity and specificity of PQ during tests at the NCJDRSU were 92% and 100%, respectively. Using these 63 CSF samples, the agreement between the two RT-QuIC generations at DRCPD and NCJDRSU prion laboratories was 100%. Full article

Early detection of cis phosphorylated tau (cis P-tau) may help as an effective treatment to control the progression of Alzheimer’s disease (AD). Recently, we introduced for the first time a monoclonal antibody (mAb) with high affinity against cis P-tau. In this study, the cis P-tau mAb was utilized to develop a label-free immunosensor. The antibody was immobilized onto a gold electrode and the electrochemical responses to the analyte were acquired by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The immunosensor was capable of selective detection of cis P-tau among non-specific targets like trans P-tau and major plasma proteins. A wide concentration range (10 × 10⁻¹⁴ M–3.0 × 10⁻⁹ M) of cis P-tau was measured in PBS and human serum matrices with a limit of detection of 0.02 and 0.05 pM, respectively. Clinical applicability of the immunosensor was suggested by its long-term storage stability and successful detection of cis P-tau in real samples of cerebrospinal fluid (CSF) and blood serum collected from human patients at different stages of AD. These results suggest that this simple immunosensor may find great application in clinical settings for early detection of AD which is an unmet urgent need in today’s healthcare services. Full article

To improve the storage and transport of clinical specimens for the diagnosis of Neisseria meningitidis (Nm) infections in resource-limited settings, we have evaluated the performance of dried blood spot (DBS) and dried cerebrospinal fluid spot (DCS) assays. DBS and DCS were prepared on filter paper from liquid specimens previously tested for Nm in the United Kingdom. Nm was detected and genogrouped by real-time PCR performed on crude genomic DNA extracted from the DBS (n = 226) and DCS (n = 226) specimens. Targeted whole-genome sequencing was performed on a subset of specimens, DBS (n = 4) and DCS (n = 6). The overall agreement between the analysis of liquid and dried specimens was (94.2%; 95% CI 90.8–96.7) for blood and (96.4%; 95% CI 93.5–98.0) for cerebrospinal fluid. Relative to liquid specimens as the reference, the DBS and DCS assays had sensitivities of (89.1%; 95% CI 82.7–93.8) and (94.2%; 95% CI 88.9–97.5), respectively, and both assays had specificities above 98%. A genogroup was identified by dried specimen analysis for 81.9% of the confirmed meningococcal infections. Near full-length Nm genome sequences (>86%) were obtained for all ten specimens tested which allowed determination of the sequence type, clonal complex, presence of antimicrobial resistance and other meningococcal genotyping. Dried blood and CSF filter spot assays offer a practical alternative to liquid specimens for the molecular and genomic characterisation of invasive meningococcal diseases in low-resource settings. Full article

Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs. Full article