Novel P2X7 Antagonist Ameliorates the Early Phase of ALS Disease and Decreases Inflammation and Autophagy in SOD1-G93A Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no effective treatment. The Hepatocyte Growth Factor/Scatter Factor (HGF/SF), through its receptor MET, is one of the most potent survival-promoting factors for motor neurons (MN) and is known as a modulator of immune cell function. We recently developed a novel recombinant MET agonist optimized for therapy, designated K1K1. K1K1 was ten times more potent than HGF/SF in preventing MN loss in an in vitro model of ALS. Treatments with K1K1 delayed the onset of muscular impairment and reduced MN loss and skeletal muscle denervation of superoxide dismutase 1 G93A (SOD1G93A) mice. This effect was associated with increased levels of phospho-extracellular signal-related kinase (pERK) in the spinal cord and sciatic nerves and the activation of non-myelinating Schwann cells. Moreover, reduced activated microglia and astroglia, lower T cells infiltration and increased interleukin 4 (IL4) levels were found in the lumbar spinal cord of K1K1 treated mice. K1K1 treatment also prevented the infiltration of T cells in skeletal muscle of SOD1G93A mice. All these protective effects were lost on long-term treatment suggesting a mechanism of drug tolerance. These data provide a rational justification for further exploring the long-term loss of K1K1 efficacy in the perspective of providing a potential treatment for ALS.

Multiple intracerebroventricular injections of human umbilical cord mesenchymal stem cells delay motor neurons loss but not disease progression of SOD1G93A mice.

Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1ß) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression.

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

ALS mouse model SOD1G93A displays early pathology of sensory small fibers associated to accumulation of a neurotoxic splice variant of peripherin.

Growing evidence suggests that amyotrophic lateral sclerosis (ALS) is a multisystem neurodegenerative disease that primarily affects motor neurons and, though less evidently, other neuronal systems. About 75% of sporadic and familial ALS patients show a subclinical degeneration of small-diameter fibers, as measured by loss of intraepidermal nerve fibers (IENFs), but the underlying biological causes are unknown. Small-diameter fibers are derived from small-diameter sensory neurons, located in dorsal root ganglia (DRG), whose biochemical hallmark is the expression of type III intermediate filament peripherin. We tested here the hypothesis that small-diameter DRG neurons of ALS mouse model SOD1(G93A)suffer from axonal stress and investigated the underlying molecular mechanism. We found that SOD1(G93A)mice display small fiber pathology, as measured by IENF loss, which precedes the onset of the disease. In vitro small-diameter DRG neurons of SOD1(G93A)mice show axonal stress features and accumulation of a peripherin splice variant, named peripherin56, which causes axonal stress through disassembling light and medium neurofilament subunits (NFL and NFM, respectively). Our findings first demonstrate that small-diameter DRG neurons of the ALS mouse model SOD1(G93A)display axonal stress in vitro and in vivo, thus sustaining the hypothesis that the effects of ALS disease spread beyond motor neurons. These results suggest a molecular mechanism for the small fiber pathology found in ALS patients. Finally, our data agree with previous findings, suggesting a key role of peripherin in the ALS pathogenesis, thus highlighting that DRG neurons mirror some dysfunctions found in motor neurons.

New Insights on the Mechanisms of Disease Course Variability in ALS from Mutant SOD1 Mouse Models.

Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous disease in terms of progression rate and survival. This is probably one of the reasons for the failure of many clinical trials and the lack of effective therapies. Similar variability is also seen in SOD1(G93A) mouse models based on their genetic background. For example, when the SOD1(G93A) transgene is expressed in C57BL6 background the phenotype is mild with slower disease progression than in the 129Sv mice expressing the same amount of transgene but showing faster progression and shorter lifespan. This review summarizes and discusses data obtained from the analysis of these two mouse models under different aspects such as the motor phenotype, neuropathological alterations in the central nervous system (CNS) and peripheral nervous system (PNS) and the motor neuron autonomous and non-cell autonomous mechanisms with the aim of finding elements to explain the different rates of disease progression. We also discuss the identification of promising prognostic biomarkers by comparative analysis of the two ALS mouse models. This analysis might possibly suggest new strategies for effective therapeutic intervention in ALS to slow significantly or even block the course of the disease.

Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model.

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1ß production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.