Astrocyte adenosine deaminase loss increases motor neuron toxicity in amyotrophic lateral sclerosis.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten.

Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease, characterised by progressive failure of the neuromuscular system. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS and frontotemporal dementia (FTD). To date, the balance of evidence indicates that the (G4C2)n repeat causes toxicity and neurodegeneration via a gain-of-toxic function mechanism; either through direct RNA toxicity or through the production of toxic aggregating dipeptide repeat proteins. Here, we have generated a stable and isogenic motor neuronal NSC34 cell model with inducible expression of a (G4C2)102 repeat, to investigate the gain-of-toxic function mechanisms. The expression of the (G4C2)102 repeat produces RNA foci and also undergoes RAN translation. In addition, the expression of the (G4C2)102 repeat shows cellular toxicity. Through comparison of transcriptomic data from the cellular model with laser-captured spinal motor neurons from C9ORF72-ALS cases, we also demonstrate that the PI3K/Akt cell survival signalling pathway is dysregulated in both systems. Furthermore, partial knockdown of Pten rescues the toxicity observed in the NSC34 (G4C2)102 cellular gain-of-toxic function model of C9ORF72-ALS. Our data indicate that PTEN may provide a potential therapeutic target to ameliorate toxic effects of the (G4C2)n repeat.

Invited review: decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art.

Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and noncoding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may coexist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function. Genetic mutations that cause neurodegenerative disorders disrupt healthy gene expression at diverse levels, from chromatin remodelling, transcription, splicing, through to axonal transport and repeat-associated non-ATG (RAN) translation. We address neurodegeneration in repeat expansion disorders [Huntington's disease, spinocerebellar ataxias, C9ORF72-related amyotrophic lateral sclerosis (ALS)] and in diseases caused by deletions or point mutations (spinal muscular atrophy, most subtypes of familial ALS). Some neurodegenerative disorders exhibit broad dysregulation of gene expression with the synthesis of hundreds to thousands of abnormal messenger RNA (mRNA) molecules. However, the number and identity of aberrant mRNAs that are translated into proteins - and how these lead to neurodegeneration - remain unknown. The field of RNA biology research faces the challenge of identifying pathophysiological events of dysregulated gene expression. In conclusion, we discuss current research limitations and future directions to improve our characterization of pathological mechanisms that trigger disease onset and progression.

Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy.

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ (2), p < 0.00001) but not sense foci (χ (2), p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.

A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery.

Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable high-throughput human cell models. Moreover, the ability to assess how astrocytic support of motor neurons is influenced by drug treatment or nutritional supplementation has been hampered by the lack of robust methodology. We have developed a high-throughput astrocyte motor neuron co-culture assay, which, by using Hb9-GFP+ motor neurons enables researchers to assess how ALS affects the ability of astrocytes to support motor neurons in 384-well plates. Moreover, astrocyte function can be manipulated by nutritional supplementation or drug treatment to identify possible therapeutic targets.

Micro-RNAs secreted through astrocyte-derived extracellular vesicles cause neuronal network degeneration in C9orf72 ALS.

BACKGROUND: Astrocytes regulate neuronal function, synaptic formation and maintenance partly through secreted extracellular vesicles (EVs). In amyotrophic lateral sclerosis (ALS) astrocytes display a toxic phenotype that contributes to motor neuron (MN) degeneration. METHODS: We used human induced astrocytes (iAstrocytes) from 3 ALS patients carrying C9orf72 mutations and 3 non-affected donors to investigate the role of astrocyte-derived EVs (ADEVs) in ALS astrocyte toxicity. ADEVs were isolated from iAstrocyte conditioned medium via ultracentrifugation and resuspended in fresh astrocyte medium before testing ADEV impact on HB9-GFP+ mouse motor neurons (Hb9-GFP+ MN). We used post-mortem brain and spinal cord tissue from 3 sporadic ALS and 3 non-ALS cases for PCR analysis. FINDINGS: We report that EV formation and miRNA cargo are dysregulated in C9ORF72-ALS iAstrocytes and this affects neurite network maintenance and MN survival in vitro. In particular, we have identified downregulation of miR-494-3p, a negative regulator of semaphorin 3A (SEMA3A) and other targets involved in axonal maintenance. We show here that by restoring miR-494-3p levels through expression of an engineered miRNA mimic we can downregulate Sema3A levels in MNs and increases MN survival in vitro. Consistently, we also report lower levels of mir-494-3p in cortico-spinal tract tissue isolated from sporadic ALS donors, thus supporting the pathological importance of this pathway in MNs and its therapeutic potential. INTERPRETATION: ALS ADEVs and their miRNA cargo are involved in MN death in ALS and we have identified miR-494-3p as a potential therapeutic target. FUNDING: Thierry Latran Fondation and Academy of Medical Sciences.

SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits.

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.

MicroNeurotrophins Improve Survival in Motor Neuron-Astrocyte Co-Cultures but Do Not Improve Disease Phenotypes in a Mutant SOD1 Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease caused by loss of motor neurons. ALS patients experience rapid deterioration in muscle function with an average lifespan of 3-5 years after diagnosis. Currently, the most effective therapeutic only extends lifespan by a few months, thus highlighting the need for new and improved therapies. Neurotrophic factors (NTFs) are important for neuronal development, maintenance, and survival. NTF treatment has previously shown efficacy in pre-clinical ALS models. However, clinical trials using NTFs produced no major improvements in ALS patients, due in part to the limited blood brain barrier (BBB) penetration. In this study we assessed the potential neuroprotective effects of a novel class of compounds known as MicroNeurotrophins (MNTs). MNTs are derivatives of Dehydroepiandrosterone (DHEA), an endogenous neurosteroid that can cross the BBB and bind to tyrosine kinase receptors mimicking the pro-survival effects of NTFs. Here we sought to determine whether MNTs were neuroprotective in two different models of ALS. Our results demonstrate that BNN27 (10 µM) attenuated loss of motor neurons co-cultured with astrocytes derived from human ALS patients with SOD1 mutations via the reduction of oxidative stress. Additionally, in the G93A SOD1 mouse, BNN27 (10 mg/kg) treatment attenuated motor behavioral impairment in the paw grip endurance and rotarod tasks at postnatal day 95 in female but not male mice. In contrast, BNN27 (10 mg/kg and 50 mg/kg) treatment did not alter any other behavioral outcome or neuropathological marker in male or female mice. Lastly, BNN27 was not detected in post-mortem brain or spinal cord tissue of treated mice due to the rapid metabolism of BNN27 by mouse hepatocytes relative to human hepatocytes. Together, these findings demonstrate that BNN27 treatment failed to yield significant neuroprotective effects in the G93A SOD1 model likely due to its rapid rate of metabolism in mice.