Study of Cyclophilin A Function in Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease targeting preferentially motor neurons. Cyclophilin A (CypA) was identified as a hallmark of disease in mutant SOD1 (mSOD1) animal models of familial ALS (fALS) at a presymptomatic stage, and in sporadic (sALS) patients (Massignan 2007; Nardo 2011). Moreover, CypA was enriched in the spinal cord aggregates of mSOD1 mice and sALS patients (Basso 2009). CypA is an ubiquitous protein with multiple functions relevant to the CNS, where it is abundantly expressed. Insights into CypA function in ALS were provided via a proteomic analysis of its interacting proteins, that functionally associated CypA with different proteins networks. In particular, it extensively binds proteins regulating RNA metabolism, including several hnRNPs and TDP-43, a major disease protein in ALS. TDP-43 and CypA interact in the nucleus, in an RNA-dependent way. CypA has a key role in the stabilization of TDP-43/hnRNP A2/B1 interaction, and TDP-43-mediated HDAC6 expression regulation, properties impaired in TDP-43 ALS-mutants, possibly because of a loss-of-interaction with CypA. CypA interacts also with mSOD1, suggesting a gain-of-interaction specifically linked to fALS. Mice expressing mSOD1 and lacking CypA show increased levels of insoluble mSOD1 and hyperphosphorylated TDP-43 in the spinal cord at the onset. This thesis work shows that CypA has a protective role in ALS: as a chaperone (for mSOD1) and in maintenance of multi-protein (TDP-43/hnRNPs) complex stability. Regardless the cause of the disease, mSOD1 or alterations in TDP-43, the interaction with CypA is impaired and it is cosequestered in proteinaceous aggregates, altering its protective activities. The net effect is the formation of pathological inclusions that may lead to a compromised RNA metabolism. CypA being a key interacting partner of both mSOD1 and TDP-43 can represent the "missing link" of these two patho-mechanisms in ALS and an interesting target for therapeutic interventions

Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients (r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression

Amyotrophic Lateral Sclerosis Multiprotein Biomarkers in Peripheral Blood Mononuclear Cells

Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms

Identification of a novel agrin-dependent pathway in cell signaling and adhesion within the erythroid niche

Establishment of cell–cell adhesion is crucial in embryonic development as well as within the stem cell niches of an adult. Adhesion between macrophages and erythroblasts is required for the formation of erythroblastic islands, specialized niches where erythroblasts proliferate and differentiate to produce red blood cells throughout life. The Eph family is the largest known family of receptor tyrosine kinases (RTKs) and controls cell adhesion, migration, invasion and morphology by modulating integrin and adhesion molecule activity and by modifying the actin cytoskeleton. Here, we identify the proteoglycan agrin as a novel regulator of Eph receptor signaling and characterize a novel mechanism controlling cell–cell adhesion and red cell development within the erythroid niche. We demonstrate that agrin induces clustering and activation of EphB1 receptors on developing erythroblasts, leading to the activation of α5β1 integrins. In agreement, agrin knockout mice display severe anemia owing to defective adhesion to macrophages and impaired maturation of erythroid cells. These results position agrin-EphB1 as a novel key signaling couple regulating cell adhesion and erythropoiesis

Tauroursodeoxycholic acid in patients with amyotrophic lateral sclerosis: The TUDCA-ALS trial protocol

Background: Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative rare disease that affects motor neurons in the brain, brainstem, and spinal cord, resulting in progressive weakness and atrophy of voluntary skeletal muscles. Although much has been achieved in understanding the disease pathogenesis, treatment options are limited, and in Europe, riluzole is the only approved drug. Recently, some other drugs showed minor effects. Methods: The TUDCA-ALS trial is a phase III, multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial. The study aims to enroll 320 patients in 25 centers across seven countries in Europe. Enrolled patients are randomized to one of two treatment arms: TUDCA or identical placebo by oral route. The study measures disease progression during the treatment period and compares it to natural progression during a no-treatment run-in phase. Clinical data and specific biomarkers are measured during the trial. The study is coordinated by a consortium composed of leading European ALS centers. Conclusion: This trial is aimed to determine whether TUDCA has a disease-modifying activity in ALS. Demonstration of TUDCA efficacy, combined with the validation of new biomarkers, could advance ALS patient care