TRANSCRIPTOME ANALYSIS OF AMOEBOID AND RAMIFIED MICROGLIA ISOLATED FROM THE CORPUS CALLOSUM OF RAT BRAIN

Ph.DDOCTOR OF PHILOSOPH

Role of activation of microglia in neurodegenerative prion disease

Prion diseases are a group of fatal neurodegenerative protein-misfolding diseases. Microglia, the resident myeloid cells found within the brain, have been shown to demonstrate a reactive morphology during the disease process with conflicting evidence for both a neurotoxic and neuroprotective role. The studies presented here aimed to investigate the role of microglia activation using transcriptomic and morphological analysis of prion disease in mice. Initially, the host immune response to prion disease was explored using a publically available mouse prion disease dataset. Re-analysis of this dataset was performed using BioLayout Express3D; a novel software tool that supports the visualisation and clustering of correlation networks. Disease-associated genes up-regulated during the later stages of infection were present in two main clusters. The cellular origin of these genes was explored by examining their expression in a dataset comprised of pure populations of cells. This demonstrated that the primary cluster of up-regulated transcripts encompassed genes expressed mainly by microglia and to a lesser extent astrocytes and neurons. The secondary cluster comprised almost exclusively of interferon response genes. The conclusions of these analyses were different from those of the original study that suggested disease-associated genes were primarily neuronal in origin. Mouse models of prion disease were established by infecting a novel line of BALB/cJ inbred mice, expressing EGFP under control of a myeloid specific Csf1r promoter, with the 79A prion strain. Quantification of the morphological changes of EGFP expressing microglia suggested the cells accumulated in the medulla at sites of early misfolded protein deposition with minimal change in their overall appearance. An activated microglia morphology was not observed until protein deposition was extensive. Isolation of EGFP expressing microglia was performed for transcriptome analysis. The vast majority of disease associated genes demonstrated increased expression at the onset of clinical symptoms. The gene list was found to be highly enriched for genes associated with an innate immune response regulated by the NFκB signalling cascade. Also highly enriched were processes associated with protein translation, energy production and stress response. These data suggest a high metabolic load is burdened by proliferating microglia; and as part of a response which is strikingly more pro-inflammatory in nature than has previously been attributed to the microglia phenotype within prion disease. As an active contributor to normal homeostasis, microglia are more than just innate immune surveillance and are now considered an integral component in both the healthy and diseased brain. The ramifications of activation toward the microglia phenotype shown here will have direct and potentially cytotoxic influence on neighbouring microglia and other brain cell types implying microglia as major contributors to the neurotoxic environment found within the CNS during prion disease. Furthermore the identification of genes associated with metabolism offer many intriguing possibilities for manipulating the activity of microglia in pre-clinical therapeutic intervention

A Molecular Mechanism for Endocytic Recycling of the M5 Muscarinic Acetylcholine Receptor

Muscarinic acetylcholine receptors (MRs), a family of five G protein-coupled receptors (GPCRs), play an essential role in the regulation of mammalian physiology. In the brain, MR-mediated neurotransmission is required for the control of movement and motivated behavior by the basal ganglia, and MR dysfunction may contribute to schizophrenia, Alzheimerʼs disease, and motor disorders. Functional studies of the muscarinic receptors have been hampered by a lack of selective pharmacology, poor receptor immunoreactivity and a wide, overlapping pattern of expression. MRs are characterized by the presence of a large third intracellular loop domain (i3), the sequence of which is divergent between MR subtypes. The i3 is known to determine signaling and trafficking characteristics of GPCRs by binding to defined subsets of regulatory and effector proteins. In an effort to discover novel, subtype-specific muscarinic receptor regulatory mechanisms, we performed yeast two-hybrid proteinprotein interaction screens with the five MR i3 regions. An interaction between M5 and the Arf GAP protein AGAP1 was detected, and was observed to be specific to the M5 subtype. This interaction was confirmed in vitro, and was shown to mediate the binding of the AP-3 adaptor complex to the M5 i3. Immunocytochemical and live cell imaging of primary rat hippocampal neurons revealed co-localization of M5 and AGAP1- or AP-3- positive vesicles after treatment with a muscarinic agonist. Activity-induced receptor trafficking studies demonstrated that interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M5 in neurons, the lack of which resulted in downregulation of cell surface receptor density. M5 has been shown to be expressed in the dopaminergic neurons of the ventral midbrain and to function in the presynaptic modulation of dopamine release in the striatum. Results from dopamine release studies suggest that the abrogation of AGAP1-mediated recycling decreases the magnitude of presynaptic M5-mediated release potentiation. Our study demonstrates a novel, neuronspecific trafficking function for AGAP1 and AP-3, and suggests the presence of a previously unknown receptor recycling pathway that may underlie mechanisms of sustained sensitivity of GPCRs

Unraveling the molecular mechanism underlying ALS-linked astrocyte toxicity for motor neurons

Mutations in superoxide dismutase-1 (SOD1) cause a familial form of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder. Transgenic mutant SOD1 rodents capture the hallmarks of this disease, which is characterized by a progressive loss of motor neurons. Studies in chimeric and conditional transgenic mutant SOD1 mice indicate that non-neuronal cells, such as astrocytes, play an important role in motor neuron degeneration. Consistent with this non-cell autonomous scenario are the demonstrations that wild-type primary and embryonic stem cell-derived motor neurons selectively degenerate when cultured in the presence of either mutant SOD1-expressing astrocytes or medium conditioned with such mutant astrocytes. The work in this thesis rests on the use of an unbiased genomic strategy that combines RNA-Seq and "reverse gene engineering" algorithms in an attempt to decipher the molecular underpinnings of motor neuron degeneration caused by mutant astrocytes. To allow such analyses, first, mutant SOD1-induced toxicity on purified embryonic stem cell-derived motor neurons was validated and characterized. This was followed by the validation of signaling pathways identified by bioinformatics in purified embryonic stem cell-derived motor neurons, using both pharmacological and genetic techniques, leading to the discovery that nuclear factor kappa B (NF-κB) is instrumental in the demise of motor neurons exposed to mutant astrocytes in vitro. These findings demonstrate the usefulness of this novel genomic approach to study neurodegeneration and to point to NF-κB as a potential valuable therapeutic target for ALS

The role of TREM2 in neurodegeneration

Introduction: Alzheimer’s disease (AD) is the most common neurodegenerative disease and has a high prevalence worldwide. Neuroinflammation has long been known to play a role in AD. However, the findings that several genes associated with inflammation were identified as hits in AD GWAS studies brought closer attention to neuroinflammatory mechanisms in AD. TREM2 was identified as a genetic risk factor for late onset AD with a similar odds ratio to that of APOE4. TREM2 is expressed on microglia, and has been shown to be upregulated on the microglia surrounding amyloid plaques both in human post mortem tissue and AD mouse models. In this thesis, the AD pathology, microglial phenotype, genetic inflammatory profile and proteomic profile of six TREM2 variant cases (5 R47H and 1 D87N variant) were investigated and compared to sporadic AD (SAD), familial AD (FAD) and neurologically normal control cases with the hypothesis that the TREM2 variant cases will differ from both SAD and FAD cases. Materials and Methods: Immunohistochemistry was performed on the frontal cortex, temporal cortex, hippocampus, putamen and cerebellum of SAD (n=19), FAD (n=11), TREM2 variant SAD (n=3), TREM2 variant controls (no AD pathology, n=2) and neurologically normal controls (n=6) using antibodies against Aβ, tau (AT8) and microglia (Iba1, CD68, CR3-43 and P2RY12). Microglial load/area, circularity and perimeter scores were calculated for all microglial markers. The frontal cortex was homogenised from a subset of each group and RNA and protein extracted. Nanostring’s Human Inflammation panel with their nCounter Technology was used to determine the genetic profile. The proteomic profile was assessed using label-free quantitative mass spectrometry. The pathological and proteomic profile of the presubiculum was investigated using immunohistochemistry, matrix-assisted laser desorption ionisation mass spectrometry, laser-capture microdissection and further label free quantitative mass spectrometry and compared to the neighbouring area, the entorhinal cortex to assess whether it has protective properties against neurodegeneration. Results: TREM2 variant AD cases differed from other SAD and FAD cases with a significantly increased CD68 load, more circular Iba1, CR3-43 and CD68 microglial positivity suggesting the microglia were in a phenotype more consistent with phagocytosis. Furthermore, these cases showed an increased upregulation of neuroinflammatory processes and neurodegenerative processes at the genetic and proteomic level than SAD cases. TREM2 variant controls however, showed large levels of downregulation in these processes compared to all groups. APOE genotyping identified the TREM2 variant cases correlated with the presence of the ApoE4 isoform. Investigation of the presubiculum area identified a large non-fibrillar Aβ deposit that contained significantly less NFT’s, activated microglia and N-terminally truncated Aβ peptides than in the entorhinal cortex and had an altered proteomic profile more comparable to the TREM2 variant controls than any other AD cases. Conclusions: Overall, this thesis has shown that TREM2 variant cases posess differences in microglial phenotype, genetic and proteomic expression compared to either sporadic or familial AD cases. TREM2 variant controls show altered pathology and genetic profiles compared to TREM2 variant SAD cases and it can be hypothesised that these cases may use similar mechanisms to the neuroprotection observed in the presubiuclum of AD cases. TREM2’s link to APOE and the fact that the APOE genotype lacks an ApoE4 allele in TREM2 variant controls, indicates that APOE may be exerting this change between TREM2 variants, leading us to question whether the TREM2 R47H variant is acting independently. Further investigations into these pathways and the differences between TREM2 variants that develop disease and those that don’t may lead us to the mechanisms that can be targeted for treatments

An old protein in unexpected places: alpha- and beta- globin expression in mesencephalic dopaminergic neurons and glial cells

Dopaminergic neurons (DA) are an anatomically and functionally heterogeneous group of cells involved in a wide range of neuronal network activities and behaviour. Among them, mesencephalic dopaminergic neurons (mDA) are the major source of dopamine in the brain. They present two main groups of projecting cells: the A9 neurons of the Substantia Nigra (SN) and the A10 cells of the Ventral Tegmental Area (VTA). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson\u2019s disease (PD) and the loss of DA synapses in the striatum is believed to be primary cause for the disruption of the ability to control movements (E. Hirsch et al. 1988). A10 cells constitute the mesocorticolimbic pathway playing a fundamental role in reward and attention. Their abnormal function has been linked to schizophrenia, attention deficit and addiction while they are relatively spared in PD (Meyer-Lindenberg et al. 2002). The description of the repertory of genes of mDA neurons may provide crucial information on their physiology as well as on the mechanisms of cell-type specific dysfunction. Interestingly, in previous gene expression profiling experiments, mDA cells groups presented a limited number of differentially expressed genes with A9-enriched transcripts mainly related to energy metabolism and mitochondrial function (C. Y. Chung et al. 2005; James G Greene et al. 2005). A crucial requirement for metabolically active aerobic cells is a steady supply of oxygen. To this purpose, hemoglobin-like molecules occur widely in organisms ranging form bacteria to human (Vandergon 1998). Vertebrate hemoglobin is the oxygen- and carbon dioxide-carrying protein in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Additional vertebrate heme-containing proteins with structural homology to globin chains include cytoglobin, mostly described in connective tissues (M. Schmidt et al. 2004), and neuroglobin, broadly expressed in the brain (Burmester 2000; Burmester 2004). Surprisingly, hemoglobin chains have been recently detected in non-erythroid cells including macrophages, alveolar cells, eye\u2019s lens and mesangial cells of the kidney (Dugas et al. 2006; L. Liu et al. 1999; Newton et al. 2006; Nishi et al. 2008). By a combination of different gene expression platforms with Laser Capture Microdissection (LCM), we have identified the transcripts of hemoglobin alpha, adult chain 1 (Hba-a1) and hemoglobin beta, adult chain 1 (Hbb-b1) in A9 neurons. Interestingly, Hemoglobin-ImmunoReactivity (Hb-IR) decorated the large majority of A9 cells while stained only less than 5% of A10 neurons. Furthermore, we detected hemoglobin expression in almost all oligodendrocytes as well as in cortical and hippocampal astrocytes and proved that this pattern of expression was conserved in mammals. Importantly, A9 DA neurons from human post mortem brain showed hemoglobin expression. By gene expression analysis of mouse dopaminergic neuroblastoma cell lines stably transfected with alpha- and beta-chains, we observed changes in genes involved in oxygen homeostasis as well as in oxidative phosphorylation, suggesting a link between hemoglobin and mitochondrial activity. These results open a new scenario for hemoglobin role in brain physiology and in PD pathogenesis

Étude du rôle physiologique et pathologique de la famille miR-132/212 dans le cerveau

La maladie d'Alzheimer (MA) est la forme de démence la plus fréquente dans le monde. Au niveau microscopique, le cerveau des patients atteints par la MA présente deux principales caractéristiques pathologiques : les plaques amyloïdes, constituées d'agrégats du peptide Aβ (Amyloïde Bêta), et les dégénérescences neurofibrillaires, formées par des agrégats de la protéine Tau anormalement hyperphosphorylée. Parmi les facteurs endogènes qui pourraient participer à la progression de la MA, il y a les microARNs (miRs). Les miRs sont des petits ARNs non codants qui régulent l’expression de gènes cibles au niveau post-transcriptionnel. En particulier, la famille miR-132/212 est fortement régulée à la baisse dans le cerveau des patients atteints de la MA. Des études précédentes ont démontré que, chez la souris 3xTg-AD, un modèle de la MA, la délétion génétique de la famille miR-132/212 conduit à une augmentation de la phosphorylation et de l’agrégation de la protéine Tau, les deux mécanismes présumés à la base de la formation des dégénérescences neurofibrillaires. En dehors de son rôle dans la MA, la famille miR-132/212 est également impliquée dans plusieurs troubles neurologiques. Notamment, son niveau d’expression est dérégulé dans d’autres pathologies neurodégénératives, telles que la démence fronto-temporale et la maladie de Parkinson. Il est donc possible que la famille miR-132/212 contribue au processus neurodégénératif de ces pathologies. Dans ce contexte, les travaux présentés visent à étudier le rôle de la famille miR132/212 dans la MA et, plus généralement, dans le cerveau. Tout d’abord, puisque la famille miR-132/212 a déjà un rôle connu dans la formation des dégénérescences neurofibrillaires, nous avons évalué son implication dans la formation des plaques amyloïdes, deuxième caractéristique pathologique de la MA. Nous avons ainsi démontré que la délétion génétique de la famille miR-132/212 favorise la production du peptide Aβ et la formation de plaques amyloïdes chez le modèle murin 3xTg-AD. En utilisant une approche d’ARN-Seq et de bio-informatique, nous avons identifié des gènes faisant partie du réseau de la famille miR-132/212 qui ont des rôles dans la régulation du métabolisme de l'Aβ, y compris Tau, Mapk et Sirt1. En accord avec ces résultats, nous avons montré que la modulation du miR-132, ou de sa cible Sirt1, peut réguler directement la production d’Aβ dans les cellules. Finalement, nous avons démontré que les niveaux de la famille miR-132/212 corrèlent avec la quantité des plaques amyloïdes chez l'Homme. Ensuite, afin d’élucider le rôle de la famille miR-132/212 dans le cerveau, nous nous sommes concentrés sur l’identification de cibles régulées par cette dernière. Dans un premier temps, cette analyse a été conduite dans plusieurs modèles cellulaires in vitro, dans lesquels le rôle du miR-132, un des deux composants de la famille, a été spécifiquement étudié. Dans ce contexte, nous avons démontré que les cibles régulées par le miR-132 sont peu nombreuses et spécifiques au type cellulaire considéré. Dans un deuxième temps, l’analyse d’identification des cibles a été conduite dans un modèle de souris de délétion conditionnelle pour la famille miR-132/212 que nous avons spécifiquement généré. Nous avons ainsi caractérisé des cibles et des réseaux moléculaires modulés par la famille miR-132/212 dans ce modèle. Pris ensemble, ces résultats suggèrent que i) Le réseau de la famille miR-132/212, dont Sirt1 et probablement d'autres gènes cibles, participe à la production du peptide Aβ et la formation de plaques amyloïdes dans la MA ; ii) Même si le miR-132 peut potentiellement cibler un grand nombre de gènes simultanément, son ciblage est sélectif et spécifique au contexte cellulaire étudié. Enfin, les résultats obtenus mettent en évidence un ensemble de nouvelles cibles et de voies de signalisation régulées par la famille miR-132/212. En conclusion, ces travaux contribuent à l'avancement des connaissances du rôle physiologique et pathologique de la famille miR-132/212 dans le cerveau.Alzheimer's disease (AD) is the most common form of dementia in the world. At the microscopic level, two main pathological features characterize the brain of AD patients: amyloid plaques, consisting of aggregates of the Aβ (Amyloid Beta) peptide, and neurofibrillary tangles, formed by aggregates of abnormally hyperphosphorylated Tau protein. Endogenous factors that may be involved in the progression of AD include microRNAs (miRs). MiRs are small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level. In particular, the miR-132/212 family is strongly downregulated in the brain of AD patients. Previous studies have shown that in the 3xTg-AD mouse model of AD, the genetic deletion of the miR-132/212 family leads to an increase in phosphorylation and aggregation of Tau protein, two mechanisms leading to the formation of neurofibrillary tangles. Apart from its role in AD, the miR-132/212 family is also involved in several neurological disorders. In particular, its level of expression is deregulated in other neurodegenerative pathologies, such as frontotemporal dementia and Parkinson's disease. It is therefore possible that the miR-132/212 family contributes to the neurodegenerative process of these pathologies. In this context, the work presented aims to study the role of the miR-132/212 family in AD and, more generally, in the brain. First of all, since the miR-132/212 family already has a known role in the formation of neurofibrillary tangles, we wanted to evaluate its involvement in the formation of the other major pathological feature of AD: the amyloid plaques. We have demonstrated that the genetic deletion of the miR-132/212 family promotes Aβ production and amyloid plaque formation in the 3xTg-AD mice. Using RNA-Seq and bioinformatics, we identified genes of the miR-132/212 network with documented roles in the regulation of Aβ metabolism, including Tau, mapk, and sirt1. Consistent with these findings, we show that the modulation of miR-132, or its target sirt1, can directly regulate Aβ production in cells. Finally, we have shown that miR-132/212 levels correlate with the amount of amyloid plaques in humans. Then, in order to elucidate the role of the miR-132/212 family in the brain, we focused on identifying targets regulated by the miR-132/212 family. In a first step, this analysis was conducted in several in vitro cell models, in which the role of miR-132, one of two components of the family, was specifically studied. In this context, we have demonstrated that the targets regulated by miR-132 are few and specific to the cell type considered. In a second step, the target identification analysis was conducted in a conditional knockout mouse model for the miR-132/212 family that we specifically generated. We have therefore characterized the molecular targets and networks modulated by the miR-132/212 family in this model. Taken together, these results suggest that i) miR-132/212 network, including Sirt1 and likely other target genes, contributes to abnormal Aβ metabolism and senile plaque deposition in AD; ii) Although miR-132 can potentially target a large number of genes simultaneously, its targeting is selective and specific to the cellular context studied. Finally, the results obtained highlight a set of new targets and signalling pathways regulated by the miR-132/212 family. In conclusion, this work contributes to the advancement of the knowledge of the physiological and pathological role of the miR-132/212 family in the brain

Autoantibody Profiling of Cerebrospinal Fluid From Affective Disorders and Schizophrenia Patients by Phage Display: Method Optimization and Evaluation of Selected Autoantigens As Candidate Biomarkers

Immunoproteomics approach applied on affective disorder and schizophrenia patient CSF samples yielded a number of candidate autoantigens. An overall analysis of those candidates revealed that impairment of myelination might have a role in etiology of such psychiatric disorders. Validations were based on phage ELISA that is applied on the most prominent autoantigen candidate obtained from phage display selection. In parallel, a conventional ELISA by recombinant peptide rather then purified phage, helped reproduce the results that imply a tendency of autoimmune reactivity in patient CSF samples compared to controls. Comparison of these results with the closest family members of the candidate protein mapping to the same N-terminal sequence revealed that, the sequence obtained by phage display is more antigenic than its homologues. Genomic expression profile of selected candidate and three other functionally related schizophrenia susceptibility genes were studied on mRNA in brain and spinal cord of mice, from postnatal day one until postnatal day forty-two. We could observe patterns of expression that may suggest a possible interplay of candidate proteins in the relevant neuronal maintenance mechanism. Our approach using cDNA phage display used in the identification of autoantigens in inflammatory and autoimmune diseases of the CNS is quite feasible despite several drawbacks like high false-positive number. Finally, our results may raise interest on myelination related proteins that may have roles in schizoaffective symptoms. However, strictly more effort has to be implemented to confirm and get more insight on our findings, for example developing mouse models and studying a broader range of serum and CSF samples

Gene Expression Profiling of Peripheral Tissues in Amyotrophic Lateral Sclerosis

Background: Amyotrophic Lateral Sclerosis, in which cortical and spinal motor neurons degenerate, is a late onset neurodegenerative condition that accounts for ~1 in 400 UK deaths, typically within 3-5 years from the initial manifestations of disease. It forms part of a broad spectrum of clinically, genetically as well as pathologically heterogeneous disorders that include behavioural variant frontotemporal lobar degeneration (bvFTLD). A large intronic hexanucleotide G4C2 repeat expansion of >30 copies was recently identified, in 2011, in the previously uncharacterised chromosome 9 open reading frame 72 (C9ORF72) gene which is now thought to explain up to 43% of familial ALS (~20-30% of familial FTLD) and around 7% of sporadic cases. Rationale & Hypothesis: The principle aim of the PhD was to perform gene expression profiling of peripheral tissues in ALS. In the first instance whole blood was trialled. However, this proved unreliable, owing to the shear abundance of erythrocyte derived alpha and beta haemoglobin transcripts that are contained within the sample and the variability in the efficiency of its removal using the Ambion® GLOBINClear or NuGEN Ovation® WB reduction strategies. Instead disease related changes in transcription/alternative splicing were detected in a large bank (n=820) of patient and control lymphoblastoid cell lines (LCL’s) with the main purpose of: 1) elucidating further the mechanism(s) of neurotoxicity associated with the C9ORF72 G4C2 repeat expansion and, 2) establishing within this specific genetic subtype, modifiers of a fast (<2yrs) versus slow (≥4yrs) disease progression in order to identify potential new areas of therapeutic research. Methodology: Biotinylated, sense-strand cDNA targets of ~40 -70nt were hybridized onto Human Exon 1.0ST GeneChip® Arrays. A GC-RMA normalisation procedure was carried out in Partek® Genomics Suite and differentially expressed or alternatively spliced transcripts were detected at the 5% significance level (p<0.05) with a fold-change threshold of ≥ ±1.20 applied. Findings: Overall a marginal increase in gene transcription was observed with respect to C9ORF72 (59.3%, n=650/1,096) and nonC9ORF72-related_SALS patients (63.9%, n=1,148/1,796) compared to neurologically healthy controls. DAVID enriched gene ontology terms included translation, which was specific to carriers of the G4C2 repeat, in addition to RNA processing, DNA metabolism, RNP complex biogenesis and the cell cycle which reflect more common features of the broader ALS phenotype. A number of key validation targets, including several RNA binding partners of the G4C2 repeat (FUS, RPL22, NUDT2, PURA, EIF4H and HNRNPA0/F) were subsequently confirmed in a qRT-PCR assay. Isoform A/B specific transcripts of the C9ORF72 gene, itself, were found not to be differentially expressed across the LCL’s in the ECACC discovery and replication cohorts. Conclusions: Whether pathogenicity of the G4C2 expanded allele arises as a consequence of haploinsufficiency or through an aberrant gain of function mechanism has yet to be determined; although emerging evidence favours a role of RNA toxicity. In light of this model, an up-regulation in the expression of C9ORF72 binding partners and other, RNA processing & splicing related transcripts fits with the hypothesis that the cells are attempting to compensate for the sequestration of these proteins into toxic RNA foci in the cytoplasm which leads to disruption of their normal physiological function. Small sample sizes meant limited conclusions could be drawn from the analysis of C9ORF72 specific modifiers of survival in ALS. Clinical data points towards a possible effect of gender which is supported in the literature but other factors such as correlations with expansion length would need to be considered in conducting future work