Amyotrophic Lateral Sclerosis, a Multisystem Pathology: Insights into the Role of TNF\u3b1

Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNF\u3b1 is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNF\u3b1 levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNF\u3b1 toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNF\u3b1 is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNF\u3b1 in ALS in the light of its multisystem nature

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy

Creatine kinase and progression rate in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no recognized clinical prognostic factor. Creatinine kinase (CK) increase in these patients is already described with conflicting results on prognosis and survival. In 126 ALS patients who were fast or slow disease progressors, CK levels were assayed for 16 months every 4 months in an observational case-control cohort study with prospective data collection conducted in Italy. CK was also measured at baseline in 88 CIDP patients with secondary axonal damage and in two mouse strains (129SvHSD and C57-BL) carrying the same SOD1G93A transgene expression but showing a fast (129Sv-SOD1G93A) and slow (C57-SOD1G93A) ALS progression rate. Higher CK was found in ALS slow progressors compared to fast progressors in T1, T2, T3, and T4, with a correlation with Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) scores. Higher CK was found in spinal compared to bulbar-onset patients. Transgenic and non-transgenic C57BL mice showed higher CK levels compared to 129SvHSD strain. At baseline mean CK was higher in ALS compared to CIDP. CK can predict the disease progression, with slow progressors associated with higher levels and fast progressors to lower levels, in both ALS patients and mice. CK is higher in ALS patients compared to patients with CIDP with secondary axonal damage; the higher levels of CK in slow progressors patients, but also in C57BL transgenic and non-transgenic mice designs CK as a predisposing factor for disease rate progression

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function

Tight junction protein changes in experimental autoimmune encephalomyelitis models

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is characterized by vascular changes, particularly endothelial tight junction (TJ) protein (claudin-5 and occludin) alterations. During blood-brain barrier function, the vascular wall components, endothelial cells, pericytes and perivascular astrocytes, engage in crosstalks through cell-associated molecules and soluble factors. Pericyte-associated NG2 is a large transmembrane proteoglycan participating in these interactions, as well as in the control of pericyte proliferation and migration. We have analyzed the role of NG2 on endothelial TJ arrangement in two groups of mice, wild type (WT) and homozygous NG2 null (NG2-/-), affected by MOG-induced EAE. Expression and distribution of the TJ transmembrane proteins claudin-5 and occludin were analyzed in the cerebral cortex microvessels by immunohistochemistry and laser confocal microscopy. In NG2-/-mice, most cortex vessels showed an altered, chain-like claudin-5 staining pattern with aggregates distributed irregularly along the junctional membranes. Unlike the claudin-5 changes, the occludin staining pattern appeared continuous and linear and only a few cortex microvessels showed protein clustering. These TJ protein expression results in NG2-/- mice affected by EAE were compared with our previous results on WT EAE mice sacrificed at 39 days post immunization. In WT EAE both claudin-5 and occludin appeared severely damaged but occludin changes were related to more severe disease stages. Interestingly, in NG2-/- EAE-affected mice, claudin-5 and occludin formed an apparently unaffected linear and continuous junctional staining, suggesting a compensation of TJ damage, with cerebral cortex microvessels showing a restored claudin-5 and occludin junctional pattern. Overall, these observations suggest that absence of NG2 in the brain microvessels of naïve NG2 null mice may affect the normal arrangement of TJ proteins, whereas under inflammatory stimuli these effects seem to be partly reversed

Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis

BACKGROUND:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. METHODOLOGY/PRINCIPAL FINDINGS:We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. CONCLUSION/SIGNIFICANCE:Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS

Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically

We tested the efficacy of treatment with talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in pre-symptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its efficacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective