Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

Inhibition of mannan-binding lectin associated serine protease (MASP)-2 reduces the cognitive deficits in a mouse model of severe traumatic brain injury.

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI.

Early treatment with rifaximin during epileptogenesis reverses gut alterations and reduces seizure duration in a mouse model of acquired epilepsy.

The gut microbiota is altered in epilepsy and is emerging as a potential target for new therapies. We studied the effects of rifaximin, a gastrointestinal tract-specific antibiotic, on seizures and neuropathology and on alterations in the gut and its microbiota in a mouse model of temporal lobe epilepsy (TLE). Epilepsy was induced by intra-amygdala kainate injection causing status epilepticus (SE) in C57Bl6 adult male mice. Sham mice were injected with vehicle. Two cohorts of SE mice were fed a rifaximin-supplemented diet for 21 days, starting either at 24 h post-SE (early disease stage) or at day 51 post-SE (chronic disease stage). Corresponding groups of SE mice (one each disease stage) were fed a standard (control) diet. Cortical ECoG recording was done at each disease stage (24/7) for 21 days in all SE mice to measure the number and duration of spontaneous seizures during either rifaximin treatment or control diet. Then, epileptic mice ± rifaximin and respective sham mice were sacrificed and brain, gut and feces collected. Biospecimens were used for: (i) quantitative histological analysis of the gut structural and cellular components; (ii) markers of gut inflammation and intestinal barrier integrity by RTqPCR; (iii) 16S rRNA metagenomics analysis in feces. Hippocampal neuronal cell loss was assessed in epileptic mice killed in the early disease phase. Rifaximin administered for 21 days post-SE (early disease stage) reduced seizure duration (p < 0.01) and prevented hilar mossy cells loss in the hippocampus compared to epileptic mice fed a control diet. Epileptic mice fed a control diet showed a reduction of both villus height and villus height/crypt depth ratio (p < 0.01) and a decreased number of goblet cells (p < 0.01) in the duodenum, as well as increased macrophage (Iba1)-immunostaining in the jejunum (p < 0.05), compared to respective sham mice. Rifaximin's effect on seizures was associated with a reversal of gut structural and cellular changes, except for goblet cells which remained reduced. Seizure duration in epileptic mice was negatively correlated with the number of mossy cells (p < 0.01) and with villus height/crypt depth ratio (p < 0.05). Rifaximin-treated epileptic mice also showed increased tight junctions (occludin and ZO-1, p < 0.01) and decreased TNF mRNA expression (p < 0.01) in the duodenum compared to epileptic mice fed a control diet. Rifaximin administered for 21 days in chronic epileptic mice (chronic disease stage) did not change the number or duration of seizures compared to epileptic mice fed a control diet. Chronic epileptic mice fed a control diet showed an increased crypt depth (p < 0.05) and reduced villus height/crypt depth ratio (p < 0.01) compared to respective sham mice. Rifaximin treatment did not affect these intestinal changes. At both disease stages, rifaximin modified α- and ß-diversity in epileptic and sham mice compared to respective mice fed a control diet. The microbiota composition in epileptic mice, as well as the effects of rifaximin at the phylum, family and genus levels, depended on the stage of the disease. During the early disease phase, the abundance of specific taxa was positively correlated with seizure duration in epileptic mice. In conclusion, gut-related alterations reflecting a dysfunctional state, occur during epilepsy development in a TLE mouse model. A short-term treatment with rifaximin during the early phase of the disease, reduced seizure duration and neuropathology, and reversed some intestinal changes, strengthening the therapeutic effects of gut-based therapies in epilepsy.

Effect of RNS60 in amyotrophic lateral sclerosis: a phase II multicentre, randomized, double-blind, placebo-controlled trial.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options. RNS60 is an immunomodulatory and neuroprotective investigational product that has shown efficacy in animal models of ALS and other neurodegenerative diseases. Its administration has been safe and well tolerated in ALS subjects in previous early phase trials. METHODS: This was a phase II, multicentre, randomized, double-blind, placebo-controlled, parallel-group trial. Participants diagnosed with definite, probable or probable laboratory-supported ALS were assigned to receive RNS60 or placebo administered for 24 weeks intravenously (375 ml) once a week and via nebulization (4 ml/day) on non-infusion days, followed by an additional 24 weeks off-treatment. The primary objective was to measure the effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood. Secondary objectives were to measure the effect of RNS60 on functional impairment (ALS Functional Rating Scale-Revised), a measure of self-sufficiency, respiratory function (forced vital capacity, FVC), quality of life (ALS Assessment Questionnaire-40, ALSAQ-40) and survival. Tolerability and safety were assessed. RESULTS: Seventy-four participants were assigned to RNS60 and 73 to placebo. Assessed biomarkers did not differ between arms. The mean rate of decline in FVC and the eating and drinking domain of ALSAQ-40 was slower in the RNS60 arm (FVC, difference 0.41 per week, standard error 0.16, p = 0.0101; ALSAQ-40, difference -0.19 per week, standard error 0.10, p = 0.0319). Adverse events were similar in the two arms. In a post hoc analysis, neurofilament light chain increased over time in bulbar onset placebo participants whilst remaining stable in those treated with RNS60. CONCLUSIONS: The positive effects of RNS60 on selected measures of respiratory and bulbar function warrant further investigation.

Prolonged exposure to simulated microgravity promotes stemness impairing morphological, metabolic and migratory profile of pancreatic cancer cells: a comprehensive proteomic, lipidomic and transcriptomic analysis.

BACKGROUND: The impact of the absence of gravity on cancer cells is of great interest, especially today that space is more accessible than ever. Despite advances, few and contradictory data are available mainly due to different setup, experimental design and time point analyzed. METHODS: Exploiting a Random Positioning Machine, we dissected the effects of long-term exposure to simulated microgravity (SMG) on pancreatic cancer cells performing proteomic, lipidomic and transcriptomic analysis at 1, 7 and 9 days. RESULTS: Our results indicated that SMG affects cellular morphology through a time-dependent activation of Actin-based motility via Rho and Cdc42 pathways leading to actin rearrangement, formation of 3D spheroids and enhancement of epithelial-to-mesenchymal transition. Bioinformatic analysis reveals that SMG may activates ERK5/NF-κB/IL-8 axis that triggers the expansion of cancer stem cells with an increased migratory capability. These cells, to remediate energy stress and apoptosis activation, undergo a metabolic reprogramming orchestrated by HIF-1α and PI3K/Akt pathways that upregulate glycolysis and impair ß-oxidation, suggesting a de novo synthesis of triglycerides for the membrane lipid bilayer formation. CONCLUSIONS: SMG revolutionizes tumor cell behavior and metabolism leading to the acquisition of an aggressive and metastatic stem cell-like phenotype. These results dissect the time-dependent cellular alterations induced by SMG and pave the base for altered gravity conditions as new anti-cancer technology.

Defective cyclophilin A induces TDP-43 proteinopathy: implications for amyotrophic lateral sclerosis and frontotemporal dementia.

Aggregation and cytoplasmic mislocalization of TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis and frontotemporal dementia spectrum. However, the molecular mechanism by which TDP-43 aggregates form and cause neurodegeneration remains poorly understood. Cyclophilin A, also known as peptidyl-prolyl cis-trans isomerase A (PPIA), is a foldase and molecular chaperone. We previously found that PPIA interacts with TDP-43 and governs some of its functions, and its deficiency accelerates disease in a mouse model of amyotrophic lateral sclerosis. Here we characterized PPIA knock-out mice throughout their lifespan and found that they develop a neurodegenerative disease with key behavioural features of frontotemporal dementia, marked TDP-43 pathology and late-onset motor dysfunction. In the mouse brain, deficient PPIA induces mislocalization and aggregation of the GTP-binding nuclear protein Ran, a PPIA interactor and a master regulator of nucleocytoplasmic transport, also for TDP-43. Moreover, in absence of PPIA, TDP-43 autoregulation is perturbed and TDP-43 and proteins involved in synaptic function are downregulated, leading to impairment of synaptic plasticity. Finally, we found that PPIA was downregulated in several patients with amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia, and identified a PPIA loss-of-function mutation in a patient with sporadic amyotrophic lateral sclerosis . The mutant PPIA has low stability, altered structure and impaired interaction with TDP-43. These findings strongly implicate that defective PPIA function causes TDP-43 mislocalization and dysfunction and should be considered in future therapeutic approaches.

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.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

Diagnostic and prognostic values of PBMC proteins in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease for which there are no validated biomarkers. Previous exploratory studies have identified a panel of candidate protein biomarkers in peripheral blood mononuclear cells (PBMCs) that include peptidyl-prolyl cis-trans isomerase A (PPIA), heat shock cognate protein 71 kDa (HSC70), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) and TDP-43. It has also been found that PPIA plays a key role in the assembly and dynamics of ribonucleoprotein (RNP) complexes and interacts with TDP-43. Its absence accelerates disease progression in a SOD1 mouse model of ALS, and low levels of PPIA in PBMCs are associated with early-onset ALS. However, the diagnostic and prognostic values of PPIA and the other candidate protein biomarkers have not been established. We analyzed the PBMC proteins in a well-characterized cohort of ALS patients (n=93), healthy individuals (n=104) and disease controls (n=111). We used a highly controlled sample processing procedure that implies two-step differential detergent fractionation. We found that the levels of the selected PBMC proteins in the soluble and insoluble fraction, combined, have a high discriminatory power for distinguishing ALS from controls, with PPIA, hnRNPA2B1 and TDP-43 being the proteins most closely associated with ALS. We also found a shift toward increased protein partitioning in the insoluble fraction in ALS and this correlated with a worse disease phenotype. In particular, low PPIA soluble levels were associated with six months earlier death. In conclusion, PPIA is a disease modifier with prognostic potential. PBMC proteins indicative of alterations in protein and RNA homeostasis are promising biomarkers of ALS, for diagnosis, prognosis and patient stratification.

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.

Cyclophillin A deficiency accelerates RML-induced prion disease.

Prion diseases typically involve brain deposition of abnormally folded prion protein, which is associated with activated glia and increased cytokine production. Cyclophilin A (CypA) is a ubiquitous protein with peptidyl prolyl cis-trans isomerase activity, which regulates protein folding, and can be secreted by cells in response to inflammatory stimuli. On the basis of in vitro studies, CypA was proposed to mediate glial activation during prion infection. To investigate the role of CypA in vivo, we inoculated CypA+/+, CypA+/- and CypA-/- mice with the RML prion strain, and recorded the time to onset of neurological signs and to terminal disease, and the astrocyte and microglia response at presymptomatic and symptomatic stages. Time to onset of disease and survival were significantly shorter in CypA-deficient mice than CypA-expressing controls. CypA-deficient mice had significantly greater microglial activation in the presymptomatic stage, and analysis of anti- and pro-inflammatory microglial markers indicated a shift towards a pro-inflammatory phenotype. There was no difference in astrocyte activation. This suggests that CypA contributes to dampening the pro-inflammatory microglial response during the early stage of RML-induced prion disease.

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.

A pilot trial of RNS60 in amyotrophic lateral sclerosis.

INTRODUCTION: RNS60 is a novel immune-modulatory agent that has shown neuroprotective effects in amytrophic lateral sclerosis (ALS) preclinical models. RNS60 is administered by weekly intravenous infusion and daily nebulization. The objective of this pilot open-label trial was to test the feasibility, safety, and tolerability of long-term RNS60 administration in ALS patients. METHODS: The planned treatment duration was 23 weeks and the primary outcomes were safety and tolerability. Secondary outcomes included PBR28 positron emission tomography (PET) imaging and plasma biomarkers of inflammation. RESULTS: Sixteen participants with ALS received RNS60 and 13 (81%) completed 23 weeks of RNS60 treatment. There were no serious adverse events and no participants withdrew from the trial due to drug-related adverse events. There were no significant changes in the biomarkers. DISCUSSION: Long-term RNS60 administration was safe and well-tolerated. A large, multicenter, phase II trial of RNS60 is currently enrolling participants to test the effects of RNS60 on ALS biomarkers and disease progression. Muscle Nerve 59:303-308, 2019.

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.

RNS60 exerts therapeutic effects in the SOD1 ALS mouse model through protective glia and peripheral nerve rescue.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects the motor neuromuscular system leading to complete paralysis and premature death. The multifactorial nature of ALS that involves both cell-autonomous and non-cell-autonomous processes contributes to the lack of effective therapies, usually targeted to a single pathogenic mechanism. RNS60, an experimental drug containing oxygenated nanobubbles generated by modified Taylor-Couette-Poiseuille flow with elevated oxygen pressure, has shown anti-inflammatory and neuroprotective properties in different experimental paradigms. Since RNS60 interferes with multiple cellular mechanisms known to be involved in ALS pathology, we evaluated its effect in in vitro and in vivo models of ALS. METHODS: Co-cultures of primary microglia/spinal neurons exposed to LPS and astrocytes/spinal neurons from SOD1G93A mice were used to examine the effect of RNS60 or normal saline (NS) on the selective motor neuron degeneration. Transgenic SOD1G93A mice were treated with RNS60 or NS (300 µl/mouse intraperitoneally every other day) starting at the disease onset and examined for disease progression as well as pathological and biochemical alterations. RESULTS: RNS60 protected motor neurons in in vitro paradigms and slowed the disease progression of C57BL/6-SOD1G93A mice through a significant protection of spinal motor neurons and neuromuscular junctions. This was mediated by the (i) activation of an antioxidant response and generation of an anti-inflammatory environment in the spinal cord; (ii) activation of the PI3K-Akt pro-survival pathway in the spinal cord and sciatic nerves; (iii) reduced demyelination of the sciatic nerves; and (iv) elevation of peripheral CD4+/Foxp3+ T regulatory cell numbers. RNS60 did not show the same effects in 129Sv-SOD1G93A mice, which are unable to activate a protective immune response. CONCLUSION: RNS60 demonstrated significant therapeutic efficacy in C57BL/6-SOD1G93A mice by virtue of its effects on multiple disease mechanisms in motor neurons, glial cells, and peripheral immune cells. These findings, together with the excellent clinical safety profile, make RNS60 a promising candidate for ALS therapy and support further studies to unravel its molecular mechanism of action. In addition, the differences in efficacy of RNS60 in SOD1G93A mice of different strains may be relevant for identifying potential markers to predict efficacy in clinical trials.

Peptidylprolyl isomerase A governs TARDBP function and assembly in heterogeneous nuclear ribonucleoprotein complexes.

Peptidylprolyl isomerase A (PPIA), also known as cyclophilin A, is a multifunctional protein with peptidyl-prolyl cis-trans isomerase activity. PPIA is also a translational biomarker for amyotrophic lateral sclerosis, and is enriched in aggregates isolated from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. Its normal function in the central nervous system is unknown. Here we show that PPIA is a functional interacting partner of TARDBP (also known as TDP-43). PPIA regulates expression of known TARDBP RNA targets and is necessary for the assembly of TARDBP in heterogeneous nuclear ribonucleoprotein complexes. Our data suggest that perturbation of PPIA/TARDBP interaction causes 'TDP-43' pathology. Consistent with this model, we show that the PPIA/TARDBP interaction is impaired in several pathological conditions. Moreover, PPIA depletion induces TARDBP aggregation, downregulates HDAC6, ATG7 and VCP, and accelerates disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Targeting the PPIA/TARDBP interaction may represent a novel therapeutic avenue for conditions involving TARDBP/TDP-43 pathology, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression.

Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies.

A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity.

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).

Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.