Distinct neuroinflammatory signatures exist across genetic and sporadic amyotrophic lateral sclerosis cohorts

Acknowledgements This work would not be possible without the resources of the Edinburgh Brain Bank, and the tissue donors and their families. Funding This research was funded in part by the Wellcome Trust (108890/Z/15/Z) to O.M.R., a Pathological Society of Great Britain & Ireland and Jean Shanks Foundation grant (JSPS CLSG 202002) to J.M.G. and J.O., a National Institutes of Health (NIH) grant (5-R01-NS127186-02) to J.M.G., F.M.W., and J.O., a Motor Neuron Disease (MND) Scotland grant to J.M.G. and C.R.S. (2021/MNDS/RP/8440GREG), and a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (215454/Z/19/Z) to C.R.S.Peer reviewedPublisher PD

Distinct neuroinflammatory signatures exist across genetic and sporadic ALS cohorts

Acknowledgments This research was funded in part by the Wellcome Trust (108890/Z/15/Z) to OMR, a Pathological Society and Jean Shanks Foundation grant (JSPS CLSG 202002) to JMG and JOS, an NIH grant (5-R01-NS127186-02) to JMG, FMW, and JOS, a Motor Neuron Disease (MND) Scotland grant to JMG and CRS (2021/MNDS/RP/8440GREG), and a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (215454/Z/19/Z) to CRS. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This work would not be possible without the resources of the Edinburgh Brain Bank. The authors declare no conflicts of interest.Preprin

Mechanisms of Neurodegeneration in ALS and FTD

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative conditions that share key clinical, pathologic, and genetic characteristics. Neuronal inclusions rich in the RNA binding protein TDP43 are found in the majority of ALS and FTD. Moreover, the most common cause of familial ALS and FTD is a hexanucleotide (G4C2) repeat expansion mutation within the first intron of chromosome 9 open reading frame 72, or C9orf72. Mutant C9orf72 transcripts undergo repeat associated non-AUG (RAN) translation, generating five unique dipeptide repeat proteins (DPRs) that accumulate in degenerating neurons in C9orf72-associated ALS/FTD, but their significance in disease pathogenesis remains unclear. My dissertation investigates C9orf72 RAN peptides, TDP43 deposition, and their respective contributions to neurodegeneration. My central hypothesis is that C9orf72 RAN peptides disrupt TDP43 metabolism, leading to neurodegeneration via TDP43-dependent RNA misprocessing. My thesis addresses the molecular pathways responsible for neurodegeneration in ALS and FTD. Chapter 1 reviews central features of ALS and FTD, including an overview of the proposed mechanisms of C9orf72-related neurodegeneration and aspects of TDP43 deposition. I first determined whether C9orf72 RAN peptides, and more specifically which RAN peptides, are toxic to neurons. In collaboration with Dr. Magdalena Ivanova, we synthesized short polymers corresponding to the three sense-strand C9orf72 RAN products, analyzed their structures by electron microscopy and assessed their relative toxicity when applied to rodent primary cortical neurons. In doing so, we observed unique structural features for each dipeptide that correlated with their cellular internalization and relative toxicity. This work is described in further detail in Chapter 2. I next began investigating the intrinsic properties of TDP43 that are critical for downstream neuronal toxicity in disease models. TDP43 binds thousands of transcripts, particularly UG-rich sequences, and TDP43-dependent toxicity is tightly tied to its ability to recognize RNA. Intramolecular interactions between TDP43’s RNA binding domains, mediated by a salt bridge, are necessary for maintaining specificity for UG sequences. How sequence specificity of TDP43 binding to RNA affects TDP43 accumulation and survival remain unclear. Here, I show that genetically engineered mutations disrupting the TDP43 salt bridge reduce the affinity of nucleic acid binding and eliminate recognition of its native RNA targets. These same mutations dramatically destabilize TDP43, alter nuclear localization and abrogate toxicity upon overexpression in primary neurons. High-throughput RNA sequencing and splicing analyses indicated that TDP43 accumulation predominantly affects transcripts encoding components of the ribosome and oxidative phosphorylation pathways. These studies are illustrated in Chapter 3. Chapter 4 describes relevant preliminary work with implications of a connection between the mutant C9orf72 repeat expansion and TDP43 deposition. Briefly, I demonstrate that G4C2 oligonucleotides are recognized by TDP43 variants containing salt bridge-disrupting mutations, and co-expression of G4C2 and TDP43 enhance cytoplasmic mislocalization and neuronal toxicity. Chapter 5 concludes the dissertation outlining the next steps moving forward with this work. Taken together, this dissertation uncovers novel disease pathways that can be targeted for therapy development.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147577/1/bnflores_1.pd

Autoimmune and autoinflammatory mechanisms in uveitis

The eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders

Stem cell models of C9orf72-linked Frontotemporal Dementia

The GGGGCC repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (ALS). Expanded repeat-associated toxicity from either RNA foci or dipeptide protein repeats (DPRs) as well as a loss of C9orf72 function due to haploinsufficiency, have all been described as potential pathogenic mechanisms in C9orf72-linked FTD/ALS (C9-FTD/ALS). Moreover, the C9orf72 protein has been suggested to play an important role in autophagy and lysosomal trafficking. The work presented in this thesis includes the investigation of FTD-related phenotypes and C9orf72 haploinsufficiency in C9orf72 patient iPSC-derived cortical neurons (iPSC-CNs), and the generation and study of C9orf72 knockout iPSC-CNs. C9orf72 patient iPSC-CNs exhibited: (i) sense RNA foci, (ii) poly-GP and poly-GR, (iii) C9orf72 promoter hypermethylation, (iv) reduction in C9orf72 total mRNA levels and (v) increased expression of intron1-retaining transcript, compared to control iPSC-CNs, with considerable variability between C9orf72 lines. No reduction in C9orf72 protein levels was detected in C9orf72 iPSC-CNs at 150 and 260 days in vitro. CRISPR/Cas9-mediated knockout of C9orf72 resulted in significant downregulation of autophagy-related genes ULK1, LC3B and LAMP1 in C9orf72-/- iPSC-CNs compared to isogenic C9orf72+/- and/or WT iPSC-CNs, indicative of altered basal autophagy. Moreover, preliminary findings suggest that induction of mitophagy is impaired in C9orf72-/- iPSC-CNs. Future work will be required to replicate this finding and further examine the potential role of C9orf72 in autophagy and mitophagy in iPSC-CNs and how its loss may contribute to C9-FTD/ALS

Pre-clinical investigation of carnosine’s anti-neoplastic effect on glioblastoma: uptake, signal transduction, gene expression and tumour cell metabolism

Das Glioblastom ist der häufigste maligne Tumor des zentralen Nervensystems. Trotz leitliniengerechter Therapie, bestehend aus mikrochirurgischer Resektion, Strahlentherapie und ergänzender Chemotherapie mit Temozolomid, beträgt die 2-Jahres-Überlebensrate nur ca. 17%. Daher sind dringend neue Therapieansätze erforderlich. Dem natürlich vorkommenden Dipeptid Carnosin, welches vor über 100 Jahren erstmals isoliert wurde, konnten viele physiologische Funktionen zugeschrieben werden. Zu Beginn unserer Arbeiten war bekannt, dass das Dipeptid das Wachstum von Krebszellen inhibiert, wobei die genauen Mechanismen der antineoplastischen Wirkungsweise weitgehend unbekannt waren. Die Untersuchungen im Rahmen der vorliegenden Habilitationsarbeit setzten sich mit möglichen Wirkmechanismen des Dipeptides auseinander, wobei ebenfalls Fragestellungen zur klinischen Anwendung von Carnosin bearbeitet wurden. Im ersten Abschnitt werden die Transportmechanismen von Carnosin in Glioblastom-Zellen beschrieben. Weiterhin wird die Frage beantwortet, ob das Dipeptid die biologisch aktive Verbindung ist oder ob L-Histidin von Carnosin abgespalten werden muss, um die antineoplastische Wirkung zu entfalten. Der zweite Abschnitt beschäftigt sich mit den Einflüssen von Carnosin auf die Signaltransduktion und Genexpression. Im dritten Abschnitt wird unter anderem mit einem Metabolomics-Ansatz der Stoffwechsel von Glioblastom-Zellen charakterisiert und der Einfluss von Carnosin auf diesen bestimmt. Im vierten Abschnitt wird ein neuartiges Ko-Kultur Modell zur Untersuchung von Carnosins Einfluss auf Glioblastom-Zell-Migration und Koloniebildung vorgestellt. Weiterhin untersuchten wir die möglichen Interaktionen des Dipeptides mit der Standardtherapie von Glioblastomen. Zusammenfassend zeigten wir, dass Carnosin durch drei verschiedene Transporter aufgenommen werden kann. Das Dipeptid hemmt sowohl Proliferation und Migration von Glioblastom-Zellen. Die Spaltung des Dipeptides ist für seine antineoplastische Wirkung nicht notwendig. In die Zelle aufgenommen, wirkt Carnosin inhibitorisch auf den Pentosephosphatweg. Eine mögliche Erklärung dafür lieferte die beobachte nicht-enzymatische Reaktion von Glycerinaldehyd-3-phosphat mit dem Dipeptid. Weiterhin zeigten unsere Experimente zum ersten Mal eine Carnosin-bedingte Veränderung der Histonacetylierung und eine damit einhergehende Beeinflussung der Genexpression. Da das Dipeptid den Effekt der Radio-/Chemotherapie verstärkt, sollte die Wirkung von Carnosin in einer klinischen Studie an Glioblastom-Patienten untersucht werden.Glioblastoma is the most common malignant tumour of the central nervous system. Only ~17% of patients undergoing standard therapy, including microsurgical resection, radiotherapy and adjuvant chemotherapy using temozolomide survive two years after diagnosis. Hence, new therapeutic approaches are urgently needed. The naturally occurring dipeptide carnosine was discovered more than 100 years ago. Since then, many physiological functions and beneficial effects have been ascribed to it. Previous studies demonstrated that carnosine inhibits growth of cancer cells. However, at the beginning of our investigations were the mechanisms behind carnosine’s anti-neoplastic effect mostly unknown. The present work addresses possible modes of action of carnosine and issues regarding the clinical application of the dipeptide. In the first paragraph we describe the transport mechanisms of carnosine in glioblastoma cells. Furthermore, we deal with the problem whether carnosine is the biological active compound or release of L-histidine from the dipeptide is required to deploy its anti-neoplastic effect. The second paragraph addresses the influence of carnosine on glioblastoma cell signal transduction and gene expression. In the third paragraph we characterise the metabolism of glioblastoma cells and how it is influenced by carnosine by using a metabolomics approach. The fourth paragraph introduces a novel co-culture model which allows the analysis of carnosine’s impact on glioblastoma cell migration and colony formation. Furthermore, the possible interaction of the dipeptide with the glioblastoma standard therapy is investigated. In conclusion, we demonstrated that three different transporters are capable for the uptake of carnosine in glioblastoma cells. The dipeptide inhibited in addition to proliferation also migration of glioblastoma cells. Moreover, cleavage of carnosine was not required for its anti-neoplastic effect. After taken up by the cell, carnosine inhibits the pentose phosphate pathway. The observed non-enzymatic reaction of glyceraldehyde-3-phosphate with the dipeptide could possibly explain this effect. Furthermore, our experiments showed for the first time that carnosine influences gene expression by an effect on histone acetylation. As the administration of carnosine arguments the effects of radio-/chemotherapy, we encourage the clinical evaluation of the dipeptide for glioblastoma patients

Genetic And Epigenetic Modifiers Of C9orf72 Expression In Neurodegenerative Disease

Repeat expansion mutations in the gene C9orf72 are the most common cause of the fatal neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The molecular mechanisms that contribute to these diseases are still not fully understood. In this dissertation, we explore mechanisms associated with repeat expansions in C9orf72 that alter gene expression and contribute to disease. In chapter 2, we develop a novel method of targeted DNA methylation in order to study how epigenetic changes in C9orf72 expansion carriers contribute to disease pathways. We find that C9orf72 promoter hypermethylation is sufficient to reduce gene expression and induce heterochromatin silencing of this locus in ALS patient derived cells. We also uncover a link between DNA damage repair pathways and DNA methylation where a double strand break in CpG islands can promote DNA methylation. Furthermore, we have taken advantage of these findings to develop and optimize a novel targeted DNA methylation method that utilizes homology directed repair for precise methylation editing in the absence of off-target effects. In chapter 3, we ask whether intermediate length repeat expansions in C9orf72 are associated with a different neurodegenerative disease, corticobasal degeneration (CBD). CBD shares similar clinical symptoms with Parkinson’s disease but exhibits distinct tau pathology in neurons and glial cells. We find that intermediate C9orf72 repeat expansion carriers (17-30 repeats) are three times more likely to develop CBD than those with smaller repeats. While full expansion carriers with ALS/FTD tend to have reduced C9orf72 expression, we show that intermediate expansion carriers actually have increased C9orf72 mRNA and protein levels. This increase in C9orf72 expression drives aberrant gene expression profiles in vesicle trafficking, stress response and autophagy pathways. Furthermore, we find that autophagy initiated by nutrient starvation is deficient in cells that over-express C9orf72. In sum, this thesis contributes a novel method of targeted DNA methylation to the research community and shows how DNA methylation alters expression of a disease relevant gene. This work also highlights how variable lengths of the repeat expansion in C9orf72 can lead to distinctive underlying molecular mechanisms and ultimately drive risk for different neurodegenerative diseases

Investigating the spread and toxicity of glycine-alanine dipeptides in C9orf72 ALS/FTD using Drosophila melanogaster

Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The role of repeat size in disease onset and severity in humans remains controversial. Transcripts of the expansions are translated into five dipeptide repeat (DPR) proteins. Most preclinical studies have used relatively short and tagged poly-DPR constructs to investigate DPR-mediated toxicity, and shown that poly-GR, poly-PR and, to a lesser extent, poly-GA DPRs are neurotoxic. Consequently, a major emphasis has been placed on understanding poly-GR- and poly-PR-mediated toxicity. However, poly-GA is the most abundant DPR in patient tissue. Transmission of protein aggregates may be a major driver of toxicity in neurodegeneration. In this study, I show for the first time that only poly-GA DPRs can spread trans-neuronally in vivo using the adult fly brain. Repeat length and tissue age modulate this phenomenon, and exosomes and synaptic vesicles are relevant in the extracellular release of GA DPRs. I also compared the toxicity, aggregation and cellular responses of GA100 DPRs carrying or not commonly used tags. Expression of tagged GA100 was markedly less toxic. GA100 tagged with GFP and mCherry exhibited aggregation differences and failed to cause DNA damage or proteostasis stress compared to untagged GA100 and GA100FLAG. These findings highlight the need to use untagged DPRs as controls when investigating their pathobiology. Finally, I tested the role of repeat size in modulating GA toxicity, subcellular localization, aggregation and cellular responses by comparing these in flies expressing untagged GA100, GA200 and GA400 DPRs. While aggregation propensity and proteostasis stress hold a positive correlation with repeat length, and GA400 was markedly more toxic than GA100, the latter was in turn more toxic than GA200. This highlights a non-linear correlation between repeat length and toxicity. GA100 and GA200 formed numerous puncta-like aggregates both in the soma and axons of neurons and, especially GA200, exhibited spreading, whereas GA400 resided only in somata and did not spread. Surprisingly, GA200 caused more DNA damage than GA100, but this effect was not observed upon GA400 expression. Collectively, I show that GA DPRs have a unique ability to spread in vivo, and their toxicity may have been previously underestimated by the use of short and tagged constructs. Therefore, my data support the further characterization of GA DPRs of a clinically relevant composition to develop strategies with therapeutic potential for C9orf72 mutation carriers