Theme 04 - In Vivo Experimetal Models

Background: A growing body of evidence shows disturbances in energy metabolism in amyotrophic lateral sclerosis (ALS) (1). The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism and play a role in oxidative stress response. Interestingly, selective inhibition of the PHD isoen- zyme 1 (PHD1) protects cortical neurons via metabolic rewiring in a model for stroke (2). As the major source of oxidative stress, which is known to be elevated in ALS, energy metabol- ism forms a promising target for ALS research. Objectives: This project aims to investigate the effect of target- ing cellular metabolism, via selective PHD1 ablation, in ALS. Methods: To investigate the role and therapeutic potential of PHD1 in ALS, we intercrossed PHD1 knock-out mice with SOD1- G93A mice and monitored disease progression, evaluating weight, neuromuscular junction innervation and total motor neuron count. Single-nuclei RNA-sequencing (snRNAseq) was performed on the lumbar spinal cord of early symptomatic mice to investigate the molecular mechanisms underlying the observed beneficial effect of PHD1 deletion in SOD1-G93A mice. Results and discussion: We demonstrated that genetic dele- tion of PHD1 improves muscle innervation and motor neuron integrity and extends the lifespan of SOD1-G93A mice, increasing the disease duration with 40%. Using snRNAseq, we were able to identify the different cell types present in the spinal cord of SOD1-G93A mice with and without PHD1. Gene ontology analysis of the differentially expressed genes showed that pathways related to oxidative metabolism are downregulated upon PHD1 deletion in motor neurons. Moreover, we also found a downregulation of interferon-stimulated genes in astrocytes. Strikingly some of these genes have recently been shown to be upregulated in ALS patients and models. A downregulation of these genes in astrocytes, which may be dependent or independent from the downregulation of reactive oxygen species (ROS) produc- ing metabolic pathways in motor neurons, could contribute to the PHD1-mediated neuroprotection. In conclusion, our data identify PHD1 inhibition as a novel ALS therapeutic strategy that targets both the metabolic dysregulation and interferon-driven hyperinflammatory response linked to ALS pathology. Rationale: Amyotrophic Lateral Sclerosis (ALS) is a fatal neuromuscular disorder characterized by motor neuron (MN) degeneration, muscle weakness, paralysis and respiratory fail- ure, leading to death within 2 – 5 years after diagnosis. ALS sprouting, process fragmentation and irregular distribution of varicosities. Notably, similar alterations in neuronal morphology and topology have been observed in muscle biopsies from ALS patients, indicating that the alteration of sympathetic innervation is a common aspect in ALS muscles. Consistently, alterations of Heart Rate Variability and a greater incidence of arrhythmic events were observed in ALS patients, indicating that SN morphological alterations are accompanied to cell dysfunction. Conclusions: Altogether, our results indicate that SNs are additional cell types compromised in ALS, adding a new piece to the complex puzzle of such uncurable disorder. Understanding how SNs are implicated in ALS may uncover additional, and yet underappreciated, therapeutic targets. Background: Increased levels of a peptide derived from Gpnmb in the cerebrospinal fluid (CSF) were recently associ- ated with a poor prognosis in patients affected by Amyotrophic Lateral Sclerosis (ALS). On the other hand, other studies highlighted that upregulation of Gpnmb could play a neuroprotective and immunomodulatory role. Objectives: In this study we engaged an in-depth character- ization of Gpnmb alterations in SOD1.G93A transgenic (TG) rat model of ALS and in patients, to clarify the value of Gpnmb as prognostic biomarker and to identify a precise time-window, during the disease process, suitable for suc-cessful therapeutic intervention. Methods: We applied in-situ hybridization (ISH) and immu-nohistochemistry (IHC) in the central and peripheral nervous system, coupled to the assessment of Gpnmb ectodomain (sGpnmb) in the CSF and blood of TG rats. In parallel, sGpnmb was assessed in a small cohort of ALS patients. Results and discussion: Gpnmb is mainly expressed in MNs in healthy conditions. However, in TG animals there is an early decrease of Gpnmb mRNA and protein levels in MNs and upregulation in reactive microglia after symptom onset. ISH and IHC highlighted a critical role for glial cells in the synthesis and release of sGpnmb. In parallel, we spotted a significant increase of sGpnmb in the CSF and blood of TG rats, as well as in ALS patients, when the pathology is more severe. We are currently running a preclinical proof of concept study to verify the therapeutic potential of early admin- istration of recombinant Gpnmb while monitoring GpnmbE as biomarker of target engagement. Through the use of the CRISPR/Cas9 system, we have generated the analogous FUS[R521H] variant (fus[R536H] knockin (KI) mutation in a Danio rerio (Zebrafish) model as well as a knockout model (fus [ (cid:1) / (cid:1) ]). We present data characterizing the degenerative phenotype in our adult homozygous [R536H] variant zebrafish. Comparing motor phenotypes through free swim and forced swim experiments we find at 3 years of age our homozygous fus[R536H] zebrafish display a significant motor deficit in comparison to both wildtypes (WT) and knockouts. This data is further supported by an autoregu- lation age-related deficit seen in our homozygous variants found through immunoblot experiments that show an increase of Fus protein in comparison to WT. RNA sequencing was performed on 1-year old WT, fus[R536H/R536H] and fus[ (cid:1) / (cid:1) ] spinal cords and mRNA misregulation of synaptic factors was found to be present in mutant spinal cords. Our results, supported by data in ALS-FUS literature, has led us to believe that knocking down mutant Fus in our animals will attenuate the phenotype in our mutants to the same level as our knockouts. We have generated potential fus-targeting ASOs and have shown significant knockdown through rt-PCR analysis in larvae. These ASOs will be delivered into the CNS of adult “ symptomatic ” mutant fish via novel cerebrointraven-tricular injections and phenotype regression will be analyzed. These results indicate a degenerative phenotype in our [R536H] variants that is unique from the behaviour, expression and analysis seen in our wildtype and knockout lines leaving promise for this model to be tested in different experimental therapies and to further our understanding of the biological nature of FUS-associated ALS. of of involved in the in both mutants. In we of involved transmission and in sporadic ALS Future directions of structural and functional neuromuscular junc- tion defects, testing the use of new gene-editing technologies repair these mutations. In ALS and FTLD, mislocalization and the formation of abnormally phosphorylated and ubiquitinated inclusions of the normally nuclear transactive response DNA-binding protein 43 (TDP-43) into the cytoplasm of diseased neurons, is a hallmark of the disease. as TDP- proteinopathy. TDP-43 proteinopathy also in open reading (C9orf72) G4C2 most Results: Large cytoplasmic EGFP-TDP-25 inclusions in neurons of the motor cortex, entorhinal cortex, visual cortex, primary somatosensory area, piriform area, amygdala, and olfactory bulb. EGFP-TDP-25 inclusions co-labeled with phosphorylated TDP-43, p62 and ubiquitin, recapitulating key hallmarks of TDP-43 proteinopathy. EGFP-TDP-25-C9KO mice formed a reduced number of EGFP-TDP-25 inclusions that were also smaller in size when compared to EGFP-TDP-25-WT mice. Furthermore, we found that EGFP-TDP-25-C9KO mice exhib- ited neuronal loss and mild motor phenotypes compared to EGFP-TDP-25-WT mice and EGFP mice of both genotypes. Autophagic deficits were assessed by p62 immunoreactivity and protein expression of ULK1. Expression of EGFP-TDP-25 led to increased cytoplasmic p62 puncta, exacerbated by the loss of C9orf72. We found higher levels of ULK1 in control C9KO mice when compared to WT mice. Yet this increase in ULK1 levels was not present in EGFP-TDP-25-C9KO mice, indi- cating altered or impaired autophagy response. Conclusion: Our findings demonstrate a synergistic effect of EGFP-TDP-25 expression with C9orf72 deficiency that leads to neuronal loss and motor behavioral deficits, contributing to a two-hit model that results in neurodegeneration in ALS/FTLD. Background: The genetic etiology of ALS is poorly understood despite concerted research efforts and an ever-expand- ing repertoire of molecular tools, with the genetic basis known in less than 20% of all cases (1). Unearthing new genes associated with ALS is crucial for progressing our understanding of disease mechanisms and the development of novel therapeutics. To this end, large collaborative multi- institute consortia have been working to characterize the genomes and transcriptomes of ALS patients. Separately, work in the translational homeostasis field has shown that disrupted ribosomal quality control (RQC) can lead to neuro-developmental, neurodegenerative, and motor phenotypes (2). Our previous work has shown that various mutations in an RQC gene, Nemf, recapitulates many key clinical features of ALS. To more solidly connect Nemf to ALS, we searched ALS genetic databases for instances of NEMF mutations in ALS patients. Of the handful of variants observed, we selected five to test in mice to determine if the variants were disease-causative. Objectives: Use a CRISPR-based in vivo model to determine the potential neuropathogenicity of NEMF mutations observed in ALS patients. Methods: We used CRISPR-Cas9 mutagenesis in mice to recreate five NEMF mutations observed in ALS patients. The resulting Nemf mutants were assessed for overt and histo-logical motor phenotypes consistent with an ALS-