Targeting Motor Neuron - Immune System Crosstalk to Modulate the Disease Progression in Amyotrophic Lateral Sclerosis Mouse Model

ALS is a fatal neurodegenerative disease characterised by remarked heterogeneity, which might stem from the multisystemic, non-cell-autonomous and complex nature of the disease. The early deterioration of the peripheral compartment has led to ALS being recognised as distal axonopathy, whereby muscles and nerves actively contribute to neurodegeneration. However, the contribution of the inflammatory response in the CNS starkly contrasts to the periphery, revealing its pivotal role at promoting phenomena of protection and/or toxicity. We corroborated these observations showing a higher activation of the MCP1 chemokine within MNs and peripheral compartment of C57SOD1G93A than 129SvSOD1G93A mice. Therefore, we surmised that the higher peripheral degeneration and faster disease progression of 129SvSOD1G93A mice stemmed from this defective immune response. To decipher the contribution of the peripheral immune response in ALS progression, the therapeutic potential of MCP1 was assessed. The chemokine was induced alongside the motor units of the two SOD1G93A models through a single intramuscular injection of a scAAV9 vector engineered with MCP1 (scAAV9_MCP1). The scAAV9_MCP1-mediated boosting of the immune response prevented the degeneration of the peripheral compartment whilst the chemokine induction within MNs led to a neuroprotective activity, resulting in the amelioration of the clinical phenotype in C57SOD1G93A but not 129SvSOD1G93A mice. This discrepancy pointed the nature and temporal activation of the immune response out as discriminating factors to promote the peripheral compartment regeneration and slow-down ALS progression. The analysis of ALS patients muscles validated our findings, demonstrating a direct correlation between the immune cells inflammatory fingerprint and the rate of the disease progression. These observations candidate the peripheral compartment as a primary target for the development of therapeutic interventions effective at influencing the ALS progression. Moreover, the comprehension of the MCP1 role within the motor unit of SOD1G93A mice might provide innovative evidence regarding the contribution of the immune response in ALS

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

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices

Micro-computed tomography for non-invasive evaluation of muscle atrophy in mouse models of disease.

Muscle wasting occurs during various chronic diseases and precedes death in humans as in mice. The evaluation of the degree of muscle atrophy in diseased mouse models is often overlooked since it requires the sacrifice of the animals for muscle examination or expensive instrumentation and highly qualified personnel, such as Magnetic Resonance Imaging (MRI). Very often behavioral tests for muscle strength evaluation are used as an outcome measurement in preclinical therapeutic trials. However, these tests are easy to perform serially, but not enough sensitive to detect early muscle changes during disease progression. Monitoring muscle loss in living animals could allow to perform more informative preclinical trials with a better evaluation of therapeutic benefit with respect to muscle wasting. We developed a non-invasive procedure based on micro-computed tomography (micro-CT) without contrast agents to monitor hind limb muscle wasting in mouse models of amyotrophic lateral sclerosis (ALS) and cancer cachexia: the transgenic SOD1G93A mouse and the colon adenocarcinoma C26-bearing mouse, respectively. We established the scanning procedure and the parameters to consider in the reconstructed images to calculate the Index of Muscle Mass (IMM). The coefficient of variance for the whole procedure was 2.2%. We performed longitudinally micro-CT scan of hind limbs in SOD1G93A mice at presymptomatic and symptomatic stages of the disease and calculated the IMM. We found that IMM in SOD1G93A mice was lower than age-matched controls even before symptom onset. We also detected a further decrease in IMM as disease progresses, most markedly just before disease onset. We performed the same analyses in the C26-based mouse model losing quickly body and muscle mass because of cancer cachexia. Overall, we found that the reduced muscle content detected by micro-CT mirrored the reduced muscle weight in both disease models. We developed a fast, precise and easy-to-conduct imaging procedure to monitor hind limb muscle mass, useful in therapeutic preclinical trials but also in proof-of-principle studies to identify the onset of muscle wasting. This method could be widely applied to other disease models characterized by muscle wasting, to assist drug development and search for early biomarkers of muscle atrophy. Moreover, reducing the number of mice needed for the experiments and being less distressing are in line with the 3R principle embodied in national and international directives for animal research

The Emerging Role of the Major Histocompatibility Complex Class I in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motoneurons (MNs). The etiology of the disease is still unknown for most patients with sporadic ALS, while in 5–10% of the familial cases, several gene mutations have been linked to the disease. Mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1), reproducing in animal models a pathological scenario similar to that found in ALS patients, have allowed for the identification of mechanisms relevant to the ALS pathogenesis. Among them, neuroinflammation mediated by glial cells and systemic immune activation play a key role in the progression of the disease, through mechanisms that can be either neuroprotective or neurodetrimental depending on the type of cells and the MN compartment involved. In this review, we will examine and discuss the involvement of major histocompatibility complex class I (MHCI) in ALS concerning its function in the adaptive immunity and its role in modulating the neural plasticity in the central and peripheral nervous system. The evidence indicates that the overexpression of MHCI into MNs protect them from astrocytes’ toxicity in the central nervous system (CNS) and promote the removal of degenerating motor axons accelerating collateral reinnervation of muscles

Micro-CT evaluation of the hind limb muscle mass and definition of the index of muscle mass (IMM).

<p>(A) Mice were placed prone on the micro-CT bed leaving the hind limbs in a natural position (foot and tibia angle mean ± SD: 41° ± 6, n = 30). (B) Scanned raw images were reconstructed in 3D and analyzed using Micro View analysis software. Scale bar, 2 mm. (C) For the evaluation of the muscle mass, the following parameters from 3D reconstructed images were measured: the distance from the upper extremity of the tibia (ut) to the medial malleolus (m), defined as the length of the tibia (L), and the perpendicular distance from the half-length of the tibia to the external margin of the hind limb muscle, referred to as the thickness of the muscle (T). These measurements were assessed on the plane in which the patella (p) and the upper extremity of tibia (ut) were clearly visualized. Scale bar, 2 mm.</p

IMM correlates with hind limb muscle weight in SOD1^G93A mice as disease progresses.

<p>(A) IMM was evaluated in Ntg and longitudinally in SOD1^G93A mice at 12, 14, 17 and 20 weeks of age (n = 5 for each group). Decrease in % was reported respect to Ntg mice at 12 weeks of age. Data, expressed as mean ± SEM, were statistically significant different (p < 0.05), by one-way ANOVA, Newman-Keuls <i>post-hoc</i> test: *, <i>versus</i> Ntg; #, <i>versus</i> 12-week old SOD1^G93A mice. (B-C) A correlation analysis was done with the mean values of the IMM and of the muscle weight of TA (B) and Gastrocnemius (GC) (C) for the different experimental groups. The analysis confirmed a significant (p < 0.008 for TA, p < 0.005 for GC) direct correlation between the two type of measurements with r² = 0.9293 and r² = 0.9502 for TA and GC respectively. Data are expressed as mean ± SEM.</p

SOD1^G93A mice have a lower hind limb muscle mass than Ntg controls already at a presymptomatic stage of the disease.

<p>(A-B) Grip strength as latency to fall (sec) and body weights (g) in Ntg and SOD1^G93A mice are reported from 11 to 22 weeks of age (n = 10 per group). Data are expressed as mean ± SEM. *, p < 0.05 by two-way ANOVA for repeated measures (the interaction between genotype and time was significant in A and B with p < 0.0001), Bonferroni’s post-hoc test. (C-D) The weight of TA (C) and Gastrocnemius (GC) (D) was measured in Ntg and SOD1^G93A mice at 12, 14, 17 and 20 weeks of age (n = 5 for each group). The weight of TA and GC was lower in SOD1^G93A mice than age-matched Ntg controls already at a presymptomatic stage of the disease (12 weeks of age). Muscle weight was expressed in mg. Decrease in % was reported with respect to Ntg mice at 12 weeks of age. Data (mean ± SEM) were significantly different (p < 0.05), by one-way ANOVA, Newman-Keuls <i>post-hoc</i> test: *, <i>versus</i> Ntg; #, <i>versus</i> 12-week old SOD1^G93A; °, <i>versus</i> 14-week old SOD1^G93A;§, <i>versus</i> 17-week old SOD1^G93A mice.</p