Leukocyte telomere length variability as a potential biomarker in patients with polyQ diseases

SCA1, SCA2, and SCA3 are the most common forms of SCAs among the polyglutamine disorders, which include Huntington's Disease (HD). We investigated the relationship between leukocyte telomere length (LTL) and the phenotype of SCA1, SCA2, and SCA3, comparing them with HD. The results showed that LTL was significantly reduced in SCA1 and SCA3 patients, while LTL was significantly longer in SCA2 patients. A significant negative relationship between LTL and age was observed in SCA1 but not in SCA2 subjects. LTL of SCA3 patients depend on both patient's age and disease duration. The number of CAG repeats did not affect LTL in the three SCAs. Since LTL is considered an indirect marker of an inflammatory response and oxidative damage, our data suggest that in SCA1 inflammation is present already at an early stage of disease similar to in HD, while in SCA3 inflammation and impaired antioxidative processes are associated with disease progression. Interestingly, in SCA2, contrary to SCA1 and SCA3, the length of leukocyte telomeres does not reduce with age. We have observed that SCAs and HD show a differing behavior in LTL for each subtype, which could constitute relevant biomarkers if confirmed in larger cohorts and longitudinal studies

Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue

\ua9 2024 by the authors.A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 \ub0C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies

2D sodium MRI of the human calf using half-sinc excitation pulses and compressed sensing

PURPOSE: Sodium MRI can be used to quantify tissue sodium concentration (TSC) in vivo; however, UTE sequences are required to capture the rapidly decaying signal. 2D MRI enables high in-plane resolution but typically has long TEs. Half-sinc excitation may enable UTE; however, twice as many readouts are necessary. Scan time can be minimized by reducing the number of signal averages (NSAs), but at a cost to SNR. We propose using compressed sensing (CS) to accelerate 2D half-sinc acquisitions while maintaining SNR and TSC. METHODS: Ex vivo and in vivo TSC were compared between 2D spiral sequences with full-sinc (TE = 0.73 ms, scan time ≈ 5 min) and half-sinc excitation (TE = 0.23 ms, scan time ≈ 10 min), with 150 NSAs. Ex vivo, these were compared to a reference 3D sequence (TE = 0.22 ms, scan time ≈ 24 min). To investigate shortening 2D scan times, half-sinc data was retrospectively reconstructed with fewer NSAs, comparing a nonuniform fast Fourier transform to CS. Resultant TSC and image quality were compared to reference 150 NSAs nonuniform fast Fourier transform images. RESULTS: TSC was significantly higher from half-sinc than from full-sinc acquisitions, ex vivo and in vivo. Ex vivo, half-sinc data more closely matched the reference 3D sequence, indicating improved accuracy. In silico modeling confirmed this was due to shorter TEs minimizing bias caused by relaxation differences between phantoms and tissue. CS was successfully applied to in vivo, half-sinc data, maintaining TSC and image quality (estimated SNR, edge sharpness, and qualitative metrics) with ≥50 NSAs. CONCLUSION: 2D sodium MRI with half-sinc excitation and CS was validated, enabling TSC quantification with 2.25 × 2.25 mm2 resolution and scan times of ≤5 mins

NAA is a Marker of Disability in Secondary-Progressive MS: A Proton MR Spectroscopic Imaging Study

BACKGROUND AND PURPOSE: The secondary progressive phase of multiple sclerosis is characterised by disability progression due to processes that lead to neurodegeneration. Surrogate markers such as those derived from MRI are beneficial in understanding the pathophysiology that drives disease progression and its relationship to clinical disability. We undertook a 1H-MRS imaging study in a large secondary progressive MS (SPMS) cohort, to examine whether metabolic markers of brain injury are associated with measures of disability, both physical and cognitive. MATERIALS AND METHODS: A cross-sectional analysis of individuals with secondary-progressive MS was performed in 119 participants. They underwent 1H-MR spectroscopy to obtain estimated concentrations and ratios to total Cr for total NAA, mIns, Glx, and total Cho in normal-appearing WM and GM. Clinical outcome measures chosen were the following: Paced Auditory Serial Addition Test, Symbol Digit Modalities Test, Nine-Hole Peg Test, Timed 25-foot Walk Test, and the Expanded Disability Status Scale. The relationship between these neurometabolites and clinical disability measures was initially examined using Spearman rank correlations. Significant associations were then further analyzed in multiple regression models adjusting for age, sex, disease duration, T2 lesion load, normalized brain volume, and occurrence of relapses in 2 years preceding study entry. RESULTS: Significant associations, which were then confirmed by multiple linear regression, were found in normal-appearing WM for total NAA (tNAA)/total Cr (tCr) and the Nine-Hole Peg Test (ρ = 0.23; 95% CI, 0.06-0.40); tNAA and tNAA/tCr and the Paced Auditory Serial Addition Test (ρ = 0.21; 95% CI, 0.03-0.38) (ρ = 0.19; 95% CI, 0.01-0.36); mIns/tCr and the Paced Auditory Serial Addition Test, (ρ = -0.23; 95% CI, -0.39 to -0.05); and in GM for tCho and the Paced Auditory Serial Addition Test (ρ = -0.24; 95% CI, -0.40 to -0.06). No other GM or normal-appearing WM relationships were found with any metabolite, with associations found during initial correlation testing losing significance after multiple linear regression analysis. CONCLUSIONS: This study suggests that metabolic markers of neuroaxonal integrity and astrogliosis in normal-appearing WM and membrane turnover in GM may act as markers of disability in secondary-progressive MS

Challenges and perspectives of quantitative functional sodium imaging (fNaI)

Brain function has been investigated via the blood oxygenation level dependent (BOLD) effect using magnetic resonance imaging (MRI) for the past decades. Advances in sodium imaging offer the unique chance to access signal changes directly linked to sodium ions (23Na) flux across the cell membrane, which generates action potentials, hence signal transmission in the brain. During this process 23Na transiently accumulates in the intracellular space. Here we show that quantitative functional sodium imaging (fNaI) at 3T is potentially sensitive to 23Na concentration changes during finger tapping, which can be quantified in gray and white matter regions key to motor function. For the first time, we measured a 23Na concentration change of 0.54 mmol/l in the ipsilateral cerebellum, 0.46 mmol/l in the contralateral primary motor cortex (M1), 0.27 mmol/l in the corpus callosum and -11 mmol/l in the ipsilateral M1, suggesting that fNaI is sensitive to distributed functional alterations. Open issues persist on the role of the glymphatic system in maintaining 23Na homeostasis, the role of excitation and inhibition as well as volume distributions during neuronal activity. Haemodynamic and physiological signal recordings coupled to realistic models of tissue function will be critical to understand the mechanisms of such changes and contribute to meeting the overarching challenge of measuring neuronal activity in vivo

Longitudinal metabolite changes in progressive multiple sclerosis:A study of 3 potential neuroprotective treatments

Background: 1 H-magnetic resonance spectroscopy (1 H-MRS) may provide a direct index for the testing of medicines for neuroprotection and drug mechanisms in multiple sclerosis (MS) through measures of total N-acetyl-aspartate (tNAA), total creatine (tCr), myo-inositol (mIns), total-choline (tCho), and glutamate + glutamine (Glx). Neurometabolites may be associated with clinical disability with evidence that baseline neuroaxonal integrity is associated with upper limb function and processing speed in secondary progressive MS (SPMS). Purpose: To assess the effect on neurometabolites from three candidate drugs after 96-weeks as seen by 1 H-MRS and their association with clinical disability in SPMS. Study-type: Longitudinal. Population: 108 participants with SPMS randomized to receive neuroprotective drugs amiloride [mean age 55.4 (SD 7.4), 61% female], fluoxetine [55.6 (6.6), 71%], riluzole [54.6 (6.3), 68%], or placebo [54.8 (7.9), 67%]. Field strength/sequence: 3-Tesla. Chemical-shift-imaging 2D-point-resolved-spectroscopy (PRESS), 3DT1. Assessment: Brain metabolites in normal appearing white matter (NAWM) and gray matter (GM), brain volume, lesion load, nine-hole peg test (9HPT), and paced auditory serial addition test were measured at baseline and at 96-weeks. Statistical tests: Paired t-test was used to analyze metabolite changes in the placebo arm over 96-weeks. Metabolite differences between treatment arms and placebo; and associations between baseline metabolites and upper limb function/information processing speed at 96-weeks assessed using multiple linear regression models. P-value<0.05 was considered statistically significant. Results: In the placebo arm, tCho increased in GM (mean difference = -0.32 IU) but decreased in NAWM (mean difference = 0.13 IU). Compared to placebo, in the fluoxetine arm, mIns/tCr was lower (β = -0.21); in the riluzole arm, GM Glx (β = -0.25) and Glx/tCr (β = -0.29) were reduced. Baseline tNAA(β = 0.22) and tNAA/tCr (β = 0.23) in NAWM were associated with 9HPT scores at 96-weeks. Data conclusion: 1 H-MRS demonstrated altered membrane turnover over 96-weeks in the placebo group. It also distinguished changes in neuro-metabolites related to gliosis and glutaminergic transmission, due to fluoxetine and riluzole, respectively. Data show tNAA is a potential marker for upper limb function. Level of evidence: 1 TECHNICAL EFFICACY: Stage 4

Longitudinal Metabolite Changes in Progressive Multiple Sclerosis: A Study of 3 Potential Neuroprotective Treatments

BACKGROUND: 1 H-magnetic resonance spectroscopy (1 H-MRS) may provide a direct index for the testing of medicines for neuroprotection and drug mechanisms in multiple sclerosis (MS) through measures of total N-acetyl-aspartate (tNAA), total creatine (tCr), myo-inositol (mIns), total-choline (tCho), and glutamate + glutamine (Glx). Neurometabolites may be associated with clinical disability with evidence that baseline neuroaxonal integrity is associated with upper limb function and processing speed in secondary progressive MS (SPMS). PURPOSE: To assess the effect on neurometabolites from three candidate drugs after 96-weeks as seen by 1 H-MRS and their association with clinical disability in SPMS. STUDY-TYPE: Longitudinal. POPULATION: 108 participants with SPMS randomized to receive neuroprotective drugs amiloride [mean age 55.4 (SD 7.4), 61% female], fluoxetine [55.6 (6.6), 71%], riluzole [54.6 (6.3), 68%], or placebo [54.8 (7.9), 67%]. FIELD STRENGTH/SEQUENCE: 3-Tesla. Chemical-shift-imaging 2D-point-resolved-spectroscopy (PRESS), 3DT1. ASSESSMENT: Brain metabolites in normal appearing white matter (NAWM) and gray matter (GM), brain volume, lesion load, nine-hole peg test (9HPT), and paced auditory serial addition test were measured at baseline and at 96-weeks. STATISTICAL TESTS: Paired t-test was used to analyze metabolite changes in the placebo arm over 96-weeks. Metabolite differences between treatment arms and placebo; and associations between baseline metabolites and upper limb function/information processing speed at 96-weeks assessed using multiple linear regression models. P-value<0.05 was considered statistically significant. RESULTS: In the placebo arm, tCho increased in GM (mean difference = -0.32 IU) but decreased in NAWM (mean difference = 0.13 IU). Compared to placebo, in the fluoxetine arm, mIns/tCr was lower (β = -0.21); in the riluzole arm, GM Glx (β = -0.25) and Glx/tCr (β = -0.29) were reduced. Baseline tNAA(β = 0.22) and tNAA/tCr (β = 0.23) in NAWM were associated with 9HPT scores at 96-weeks. DATA CONCLUSION: 1 H-MRS demonstrated altered membrane turnover over 96-weeks in the placebo group. It also distinguished changes in neuro-metabolites related to gliosis and glutaminergic transmission, due to fluoxetine and riluzole, respectively. Data show tNAA is a potential marker for upper limb function. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 4

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

<p>Abstract</p> <p>Background</p> <p>Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.</p> <p>Methods</p> <p>Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional ¹H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH₂oxidation after 24 h pre-incubation with CMH.</p> <p>Results</p> <p>The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH₂oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.</p> <p>Conclusions</p> <p>The explorative approach of this study combined with the determination of a decreased intracellular DCFH₂oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.</p

Author Correction: Enhanced NF-κB signaling in type-2 dendritic cells at baseline predicts non-response to adalimumab in psoriasis.

Funder: Department of HealthBiologic therapies have transformed the management of psoriasis, but clinical outcome is variable leaving an unmet clinical need for predictive biomarkers of response. Here we perform in-depth immunomonitoring of blood immune cells of 67 patients with psoriasis, before and during therapy with the anti-TNF drug adalimumab, to identify immune mediators of clinical response and evaluate their predictive value. Enhanced NF-κBp65 phosphorylation, induced by TNF and LPS in type-2 dendritic cells (DC) before therapy, significantly correlates with lack of clinical response after 12 weeks of treatment. The heightened NF-κB activation is linked to increased DC maturation in vitro and frequency of IL-17+ T cells in the blood of non-responders before therapy. Moreover, lesional skin of non-responders contains higher numbers of dermal DC expressing the maturation marker CD83 and producing IL-23, and increased numbers of IL-17+ T cells. Finally, we identify and clinically validate LPS-induced NF-κBp65 phosphorylation before therapy as a predictive biomarker of non-response to adalimumab, with 100% sensitivity and 90.1% specificity in an independent cohort. Our study uncovers important molecular and cellular mediators underpinning adalimumab mechanisms of action in psoriasis and we propose a blood biomarker for predicting clinical outcome