Mitochondrial Aconitase Enzymatic Activity: A Potential Long-Term Survival Biomarker in the Blood of ALS Patients.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multisystemic, progressive, neurodegenerative disorder. Despite it being generally fatal within a period of 2-4 years, it is highly heterogeneous; as a result, survival periods may vary greatly among individual patients. Biomarkers can serve as tools for diagnosis, prognosis, indicators of therapeutic response, and future therapeutics. Free-radical-dependent mitochondrial damage is believed to play a crucial role in neurodegeneration in ALS. Mitochondrial aconitase, which is also known as aconitase 2 (Aco2), is a key Krebs cycle enzyme and is involved in the regulation of cellular metabolism and iron homeostasis. Aco2 is very sensitive to oxidative inactivation and can aggregate and accumulate in the mitochondrial matrix, causing mitochondrial dysfunction. Loss of Aco2 activity may therefore reflect increased levels of mitochondrial dysfunction due to oxidative damage and could be relevant to ALS pathogenesis. The aim of our study was to confirm changes in mitochondrial aconitase activity in peripheral blood and to determine whether such changes are dependent on, or independent of, the patient's condition and to propose the feasibility of using them as possible valid biomarkers to quantify the progression of the disease and as a predictor of individual prognosis in ALS. METHODS: We measured the Aco2 enzymatic activity in the platelets of blood samples taken from 22 controls and 26 ALS patients at different stages of disease development. We then correlated antioxidant activity with clinical and prognostic variables. RESULTS: Aco2 activity was significantly lower in the 26 ALS patients than in the 22 controls (p < 0.05). Patients with higher levels of Aco2 activity survived longer than those with lower levels (p < 0.05). Aco2 activity was also higher in patients with earlier onset (p < 0.05) and in those with predominantly upper motor neuron signs. CONCLUSIONS: Aco2 activity seems to be an independent factor that could be used in the long-term survival prognosis of ALS. Our findings suggest that blood Aco2 could be a leading candidate for use as a biomarker to improve prognosis. More studies are needed to confirm these results.

Baseline Inflammatory Status Reveals Dichotomic Immune Mechanisms Involved In Primary-Progressive Multiple Sclerosis Pathology.

Objective: To ascertain the role of inflammation in the response to ocrelizumab in primary-progressive multiple sclerosis (PPMS). Methods: Multicenter prospective study including 69 patients with PPMS who initiated ocrelizumab treatment, classified according to baseline presence [Gd+, n=16] or absence [Gd-, n=53] of gadolinium-enhancing lesions in brain MRI. Ten Gd+ (62.5%) and 41 Gd- patients (77.4%) showed non-evidence of disease activity (NEDA) defined as no disability progression or new MRI lesions after 1 year of treatment. Blood immune cell subsets were characterized by flow cytometry, serum immunoglobulins by nephelometry, and serum neurofilament light-chains (sNfL) by SIMOA. Statistical analyses were corrected with the Bonferroni formula. Results: More than 60% of patients reached NEDA after a year of treatment, regardless of their baseline characteristics. In Gd+ patients, it associated with a low repopulation rate of inflammatory B cells accompanied by a reduction of sNfL values 6 months after their first ocrelizumab dose. Patients in Gd- group also had low B cell numbers and sNfL values 6 months after initiating treatment, independent of their treatment response. In these patients, NEDA status was associated with a tolerogenic remodeling of the T and innate immune cell compartments, and with a clear increase of serum IgA levels. Conclusion: Baseline inflammation influences which immunological pathways predominate in patients with PPMS. Inflammatory B cells played a pivotal role in the Gd+ group and inflammatory T and innate immune cells in Gd- patients. B cell depletion can modulate both mechanisms.

Effect of Ocrelizumab in Blood Leukocytes of Patients With Primary Progressive MS.

OBJECTIVE: To analyze the changes induced by ocrelizumab in blood immune cells of patients with primary progressive MS (PPMS). METHODS: In this multicenter prospective study including 53 patients with PPMS who initiated ocrelizumab treatment, we determined effector, memory, and regulatory cells by flow cytometry at baseline and after 6 months of therapy. Wilcoxon matched paired tests were used to assess differences between baseline and 6 months' results. p Values were corrected using the Bonferroni test. RESULTS: Ocrelizumab reduced the numbers of naive and memory B cells (p < 0.0001) and those of B cells producing interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNFα) (p < 0.0001 in all cases). By contrast, the proportions of plasmablasts and B cells producing GM-CSF and TNFα increased significantly, suggesting the need for treatment continuation. We also observed a decrease in CD20+ T-cell numbers (p < 0.0001) and percentages (p < 0.0001), and a clear remodeling of the T-cell compartment characterized by relative increases of the naive/effector ratios in CD4+ (p = 0.002) and CD8+ (p = 0.002) T cells and relative decreases of CD4+ (p = 0.03) and CD8+ (p = 0.004) T cells producing interferon-gamma. Total monocyte numbers increased (p = 0.002), but no changes were observed in those producing inflammatory cytokines. The immunologic variations were associated with a reduction of serum neurofilament light chain (sNfL) levels (p = 0.008). The reduction was observed in patients with Gd-enhanced lesions at baseline and in Gd- patients with baseline sNfL >10 pg/mL. CONCLUSIONS: In PPMS, effector B-cell depletion changed T-cell response toward a low inflammatory profile, resulting in decreased sNfL levels.

Usefulness of lyso-globotriaosylsphingosine in dried blood spots in the differential diagnosis between multiple sclerosis and Anderson-Fabry's disease.

BACKGROUND: The presence of white mater lesions in the central nervous system forces the differential diagnosis between multiple sclerosis (MS) and Anderson-Fabry disease (FD). Due to the type of inheritance, linked to the X chromosome, the diagnosis of FD is especially difficult in women. Tissue´s deposits of globotriaosylceramide (Gb3) are characteristics for FD and the deacylated form of Gb3 (Globotriaosylsphingosine or LysoGb3) is specific for this entity. Our objective is to investigate if concentrations of plasma Lyso-Gb3 are useful for ruling out the FD in a Spanish cohort of patients with a previous diagnosis of MS. METHODS: we evaluated the α-galactosidase A enzymatic activity in 154 patients with a previous diagnosis of MS (93 women and 61 men): 103 Relapsing Remitting MS patients, 19 progressive MS patients and 32 with the clinically isolated syndrome. 116 (75% of the patients) were on MS disease modifying therapy. Enzymatic assay was completed in all cases and done on dried blood spot (DBS) samples. Subsequently the GLA gene was sequenced only in males and females who presented an enzymatic assay significantly lower than standardized controls (<50% for men and <75% for women). For subjects with GLA variants, plasma Lyso-Gb3 levels were performed by Tandem mass spectrometry from DBS, assuming a cut-off point for normality <3.5 ng/mL. RESULTS: Genetic study was carried out in 30 women and 7 men; 8 of them had non-previous described GLA variants. After a thorough clinical examination no organic disease was found in any of the classical target organs. The study of Lyso-Gb3 concentrations in DBS was lower than 3.5 ng/mL, allowing us to discharge FD in all subjects and to consider these GLA variants like non pathologic. CONCLUSIONS: Lyso-Gb3 concentration in DBS is a useful tool to rule out Fabry disease in patients with MS. A concentration of LysoGb3 < 3.5 ng/mL rules out FD.

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.

Mitochondrial complex enzyme activities and cytochrome C expression changes in multiple sclerosis.

Blood platelets have been widely proposed as biomarkers in studies of mitochondrial function and aging-related and neurodegenerative diseases. Defects in mitochondrial function were found not only in the substantia nigra of Parkinson's disease patients but also in their blood platelets. Similarly, it has also been described in the blood platelet mitochondria of Alzheimer's disease patients. To study mitochondrial aerobic metabolism function and protein expression in platelets of multiple sclerosis (MS) patients and control subjects, mitochondrial aconitase, mitochondrial superoxide dismutases 1 and 2 (SOD1 and SOD2), and respiratory complex enzyme activities in platelets of MS patients and control subjects were determined. Likewise, mitochondrial lipid peroxidation and mitochondrial SOD1 and cytochrome c expressions were investigated. Mitochondrial aconitase activity was higher in MS patients than in controls (P < 0.05). A significant increase on all respiratory complex activities in MS patients was observed (P < 0.05). Mitochondrial lipid peroxidation was significantly higher in MS patients than in controls (P < 0.05). Significant changes of cytochrome c and mitochondrial SOD1 expressions were detected (P < 0.05), with a decrease of 44 ± 5 % and an increase of 46 ± 6 %, respectively. Our study reveals that significant changes in mitochondrial aerobic metabolism function and mitochondrial SOD1 and cytochrome c expressions are produced in platelets of MS patients.

Analysis of prognostic factors associated with longitudinally extensive transverse myelitis.

OBJECTIVE: The aim of this study is to report the clinical profile and outcome of longitudinally extensive transverse myelitis (LETM). METHODS: We prospectively studied adult patients who presented with LETM from January 2008 to December 2011. Information on demographic, clinical course, magnetic resonance imaging (MRI) and outcome was collected. HLA-DRB1 genotype was compared with those of 225 normal controls and patients with MS (228) and neuromyelitis optica (NMO) (22). RESULTS: In total, 23 patients (16 female) with a median age of 44.5 years (range: 20-77 years) were included. Most (74%) had moderate-severe disability at nadir (48% non-ambulatory), normal/non-multiple sclerosis (MS) brain MRI (96%) and a median MRI cord lesion of 5 vertebral segments (range: 3-19). Laboratory analysis showed cerebrospinal fluid pleocytosis (45%), NMO-IgG (9%), antinuclear antibodies (70%), and genotype HLA-DRB1*13 (57%). The frequency of DRB1*13 genotype was higher compared with controls (p=0.002), MS (p=0.001) and NMO (p=0.003) patients. After a median follow-up of 32 months, one patient converted to MS, two had relapsing LETM with NMO-IgG, and 20 remained as idiopathic with recurrences in four (20%). Twelve (52%) patients recovered with minimal disability (Expanded Disability Status Scale (EDSS) ≤2.5) and three (13%) remained wheelchair dependent. Disability at nadir was associated with the final outcome and extension of the spinal cord lesion with risk of recurrence. Recurrence was not associated with worse outcome. CONCLUSIONS: Inflammatory LETM is mostly idiopathic with a good outcome. It includes a relatively homogenous group of patients with an overrepresentation of the HLA-DRB1*13 genotype. EDSS at nadir is a predictor of the final outcome and extension of the myelitis of the recurrence risk.

Diabetes mellitus y enfermedad cerebrovascular aguda (Ictus-ACV) / Diabetes mellitus and stroke

Se llevó a cabo un estudio prospectivo de 415 pacientes con ictus ingresados consecutivamente durante un año. Se excluyeron la isquemia transitoria y la hemorragia subaracnoidea. Se analizó edad, sexo, factores de riesgo, gravedad, mortalidad y pronóstico funcional a la semana. De los 415 pacientes 354 fueron diagnosticados de infarto cerebral y 61 de hemorragia parenquimatosa. El factor de riesgo más frecuente fue la hipertensión arterial. Padecían DM 95 pacientes. El análisis bi y multivariante determina como variables indpendientes asociadas a la DM el debut de la patología cerebrovascular en edades más jóvenes (p=0,009), la asociación a hipertensión arterial (p=0,002) y la peor calidad de vida previa (p=0,003). No se encontró mayor frecuencia de infartos lacunares entre los pacientes diabéticos. El paciente diabético es más jóven y con frecuencia asocia hipertensión. No existen diferencias en cuanto a la mortalidad y tipo de ACV. Tampoco se encontró una relación significativa con el infarto lacunar