Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.

Chemotherapy-induced neutropenia elicits metastasis formation in mice by promoting proliferation of disseminated tumor cells.

Chemotherapy is the standard of care for most malignancies. Its tumor debulking effect in adjuvant or neoadjuvant settings is unquestionable, although secondary effects have been reported that paradoxically promote metastasis. Chemotherapy affects the hematopoietic precursors leading to myelosuppression, with neutropenia being the main hematological toxicity induced by cytotoxic therapy. We used renal and lung murine tumor models metastatic to the lung to study chemotherapy-induced neutropenia (CIN) in the metastatic process. Cyclophosphamide and doxorubicin, two myelosuppressive drugs, but not cisplatin, increased the burden of artificial metastases to the lung, by reducing neutrophils. This effect was recapitulated by treatment with anti-Ly6G, the selective antibody-mediated neutrophil depletion that unleashed the formation of lung metastases in both artificial and spontaneous metastasis settings. The increased cancer dissemination was reversed by granulocyte-colony stimulating factor-mediated boosting of neutrophils in combination with chemotherapy. CIN affected the early metastatic colonization of the lung, quite likely promoting the proliferation of tumor cells extravasated into the lung at 24-72 hours. CIN did not affect the late events of the metastatic process, with established metastasis to the lung, nor was there any effect on the release of cancer cells from the primary, whose growth was, in fact, somewhat inhibited. This work suggests a role of neutrophils associated to a common cancer treatment side effect and claims a deep dive into the relationship between chemotherapy-induced neutropenia and metastasis.

Contingent intramuscular boosting of P2XR7 axis improves motor function in transgenic ALS mice.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons and severe muscle atrophy without effective treatment. Most research on the disease has been focused on studying motor neurons and supporting cells of the central nervous system. Strikingly, the recent observations have suggested that morpho-functional alterations in skeletal muscle precede motor neuron degeneration, bolstering the interest in studying muscle tissue as a potential target for the delivery of therapies. We previously showed that the systemic administration of the P2XR7 agonist, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (BzATP), enhanced the metabolism and promoted the myogenesis of new fibres in the skeletal muscles of SOD1G93A mice. Here we further corroborated this evidence showing that intramuscular administration of BzATP improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of infiltrating macrophages. The preservation of the skeletal muscle retrogradely propagated along with the motor unit, suggesting that backward signalling from the muscle could impinge on motor neuron death. In addition to providing the basis for a suitable adjunct multisystem therapeutic approach in ALS, these data point out that the muscle should be at the centre of ALS research as a target tissue to address novel therapies in combination with those oriented to the CNS.

CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis.

BACKGROUND: CXCL13 is a B and T lymphocyte chemokine that mediates neuroinflammation through its receptor CXCR5. This chemokine is highly expressed by motoneurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) SOD1G93A (mSOD1) mice during the disease, particularly in fast-progressing mice. Accordingly, in this study, we investigated the role of this chemokine in ALS. METHODS: We used in vitro and in vivo experimental paradigms derived from ALS mice and patients to investigate the expression level and distribution of CXCL13/CXCR5 axis and its role in MN death and disease progression. Moreover, we compared the levels of CXCL13 in the CSF and serum of ALS patients and controls. FINDINGS: CXCL13 and CXCR5 are overexpressed in the spinal MNs and peripheral axons in mSOD1 mice. CXCL13 inhibition in the CNS of ALS mice resulted in the exacerbation of motor impairment (n = 4/group;Mean_Diff.=27.81) and decrease survival (n = 14_Treated:19.2 ± 1.05wks, n = 17_Controls:20.2 ± 0.6wks; 95% CI: 0.4687-1.929). This was corroborated by evidence from primary spinal cultures where the inhibition or activation of CXCL13 exacerbated or prevented the MN loss. Besides, we found that CXCL13/CXCR5 axis is overexpressed in the spinal cord MNs of ALS patients, and CXCL13 levels in the CSF discriminate ALS (n = 30) from Multiple Sclerosis (n = 16) patients with a sensitivity of 97.56%. INTERPRETATION: We hypothesise that MNs activate CXCL13 signalling to attenuate CNS inflammation and prevent the neuromuscular denervation. The low levels of CXCL13 in the CSF of ALS patients might reflect the MN dysfunction, suggesting this chemokine as a potential clinical adjunct to discriminate ALS from other neurological diseases. FUNDING: Vaccinex, Inc.; Regione Lombardia (TRANS-ALS).

5'ValCAC tRNA fragment generated as part of a protective angiogenin response provides prognostic value in amyotrophic lateral sclerosis.

Loss-of-function mutations in the ribonuclease angiogenin are associated with amyotrophic lateral sclerosis. Angiogenin has been shown to cleave transfer RNAs during stress to produce 'transfer-derived stress-induced RNAs'. Stress-induced tRNA cleavage is preserved from single-celled organisms to humans indicating it represents part of a highly conserved stress response. However, to date, the role of tRNA cleavage in amyotrophic lateral sclerosis remains to be fully elucidated. To this end, we performed small RNA sequencing on a human astrocytoma cell line to identify the complete repertoire of tRNA fragments generated by angiogenin. We found that only a specific subset of tRNAs is cleaved by angiogenin and identified 5'ValCAC transfer-derived stress-induced RNA to be secreted from neural cells. 5'ValCAC was quantified in spinal cord and serum from SOD1G93A amyotrophic lateral sclerosis mouse models where we found it to be significantly elevated at symptom onset correlating with increased angiogenin expression, imbalanced protein translation initiation factors and slower disease progression. In amyotrophic lateral sclerosis patient serum samples, we found 5'ValCAC to be significantly higher in patients with slow disease progression, and interestingly, we find 5'ValCAC to hold prognostic value for amyotrophic lateral sclerosis patients. Here, we report that angiogenin cleaves a specific subset of tRNAs and provide evidence for 5'ValCAC as a prognostic biomarker in amyotrophic lateral sclerosis. We propose that increased serum 5'ValCAC levels indicate an enhanced angiogenin-mediated stress response within motor neurons that correlates with increased survival. These data suggest that the previously reported beneficial effects of angiogenin in SOD1G93A mice may result from elevated levels of 5'ValCAC transfer RNA fragment.

P2X7 activation enhances skeletal muscle metabolism and regeneration in SOD1G93A mouse model of amyotrophic lateral sclerosis.

Muscle weakness plays an important role in neuromuscular disorders comprising amyotrophic lateral sclerosis (ALS). However, it is not established whether muscle denervation originates from the motor neurons, the muscles or more likely both. Previous studies have shown that the expression of the SOD1G93A mutation in skeletal muscles causes denervation of the neuromuscular junctions, inability to regenerate and consequent atrophy, all clear symptoms of ALS. In this work, we used SOD1G93A mice, a model that best mimics some pathological features of both familial and sporadic ALS, and we investigated some biological effects induced by the activation of the P2X7 receptor in the skeletal muscles. The P2X7, belonging to the ionotropic family of purinergic receptors for extracellular ATP, is abundantly expressed in the healthy skeletal muscles, where it controls cell duplication, differentiation, regeneration or death. In particular, we evaluated whether an in vivo treatment in SOD1G93A mice with the P2X7 specific agonist 2'(3')-O-(4-Benzoylbenzoyl) adenosine5'-triphosphate (BzATP) just before the onset of a pathological neuromuscular phenotype could exert beneficial effects in the skeletal muscles. Our findings indicate that stimulation of P2X7 improves the innervation and metabolism of myofibers, moreover elicits the proliferation/differentiation of satellite cells, thus preventing the denervation atrophy of skeletal muscles in SOD1G93A mice. Overall, this study suggests that a P2X7-targeted and site-specific modulation might be a strategy to interfere with the complex multifactorial and multisystem nature of ALS.

Stimulation of P2X7 Enhances Whole Body Energy Metabolism in Mice.

The P2X7 receptor, a member of the ionotropic purinergic P2X family of extracellular ATP-gated receptors, exerts strong trophic effects when tonically activated in cells, in addition to cytotoxic effects after a sustained activation. Because of its widespread distribution, P2X7 regulates several cell- and tissue-specific physiological functions, and is involved in a number of disease conditions. A novel role has recently emerged for P2X7 in the regulation of glucose and energy metabolism. In previous work, we have demonstrated that genetic depletion, and to a lesser extent also pharmacological inhibition of P2X7, elicits a significant decrease of the whole body energy expenditure and an increase of the respiratory exchange ratio. In the present work, we have investigated the effects of P2X7 stimulation in vivo on the whole body energy metabolism. Adult mice were daily injected with the specific P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate for 1 week and subjected to indirect calorimetric analysis for 48 h. We report that 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate increases metabolic rate and O2 consumption, concomitantly decreasing respiratory rate and upregulating NADPH oxidase 2 in gastrocnemius and tibialis anterior muscles. Our results indicate a major impact on energy homeostasis and muscle metabolism by activation of P2X7.

Histaminergic transmission slows progression of amyotrophic lateral sclerosis.

BACKGROUND: Histamine is an immune modulator, neuroprotective, and remyelinating agent, beneficially acting on skeletal muscles and promoting anti-inflammatory features in amyotrophic lateral sclerosis (ALS) microglia. Drugs potentiating the endogenous release of histamine are in trial for neurological diseases, with a role not systematically investigated in ALS. Here, we examine histamine pathway associations in ALS patients and the efficacy of a histamine-mediated therapeutic strategy in ALS mice. METHODS: We adopted an integrative multi-omics approach combining gene expression profiles, copy number variants, and single nucleotide polymorphisms of ALS patients. We treated superoxide dismutase 1 (SOD1)-G93A mice that recapitulate key ALS features, with the brain-permeable histamine precursor histidine in the symptomatic phase of the disease and analysed the rescue from disease pathological signs. We examined the action of histamine in cultured SOD1-G93A motor neuron-like cells. RESULTS: We identified 13 histamine-related genes deregulated in the spinal cord of two ALS patient subgroups, among which genes involved in histamine metabolism, receptors, transport, and secretion. Some histamine-related genes overlapped with genomic regions disrupted by DNA copy number and with ALS-linked pathogenic variants. Histidine treatment in SOD1-G93A mice proved broad efficacy in ameliorating ALS features, among which most importantly lifespan, motor performance, microgliosis, muscle atrophy, and motor neurons survival in vivo and in vitro. CONCLUSIONS: Our gene set/pathway enrichment analyses and preclinical studies started at the onset of symptoms establish that histamine-related genes are modifiers in ALS, supporting their role as candidate biomarkers and therapeutic targets. We disclose a novel important role for histamine in the characterization of the multi-gene network responsible for ALS and, furthermore, in the drug development process.

Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis.

Multiple sclerosis (MS) is characterized by macrophage accumulation and inflammatory infiltrates into the CNS contributing to demyelination. Because purinergic P2X7 receptor (P2X7R) is known to be abundantly expressed on cells of the hematopoietic lineage and of the nervous system, we further investigated its phenotypic expression in MS and experimental autoimmune encephalomyelitis conditions. By quantitative reverse transcription polymerase chain reaction and flow cytometry, we analyzed the P2X7R expression in human mononuclear cells of peripheral blood from stable and acute relapsing-remitting MS phases. Human monocytes were also challenged in vitro with pro-inflammatory stimuli such as the lipopolysaccharide, or the P2X7R preferential agonist 2'(3')-O-(4 Benzoylbenzoyl)adenosine 5'-triphosphate, before evaluating P2X7R protein expression. Finally, by immunohistochemistry and immunofluorescence confocal analysis, we investigated the P2X7R expression in frontal cortex from secondary progressive MS cases. We demonstrated that P2X7R is present and inhibited on peripheral monocytes isolated from MS donors during the acute phase of the disease, moreover it is down-regulated in human monocytes after pro-inflammatory stimulation in vitro. P2X7R is instead up-regulated on astrocytes in the parenchyma of frontal cortex from secondary progressive MS patients, concomitantly with monocyte chemoattractant protein-1 chemokine, while totally absent from microglia/macrophages or oligodendrocytes, despite the occurrence of inflammatory conditions. Our results suggest that inhibition of P2X7R on monocytes and up-regulation in astrocytes might contribute to sustain inflammatory mechanisms in MS. By acquiring further knowledge about P2X7R dynamics and identifying P2X7R as a potential marker for the disease, we expect to gain insights into the molecular pathways of MS.