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In this Perspective, owing to an error in the HTML, the surname of author Alejandro López-Tobón of the LifeTime Community Working Groups consortium was indexed as ‘Tobon’ rather than ‘López-Tobón’ and the accents were missing. The HTML version of the original Perspective has been corrected; the PDF and print versions were always correct. © 2021, The Author(s).

Objective: Substantial clinical, pathological, and genetic overlap exists between amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 inclusions have been found in both ALS and FTD cases (FTD-TDP). Recently, a repeat expansion in C9orf72 was identified as the causal variant in a proportion of ALS and FTD cases. We sought to identify additional evidence for a common genetic basis for the spectrum of ALS-FTD. Methods: We used published genome-wide association studies data for 4,377 ALS patients and 13,017 controls, and 435 pathology-proven FTD-TDP cases and 1,414 controls for genotype imputation. Data were analyzed in a joint meta-analysis, by replicating topmost associated hits of one disease in the other, and by using a conservative rank products analysis, allocating equal weight to ALS and FTD-TDP sample sizes. Results: Meta-analysis identified 19 genome-wide significant single nucleotide polymorphisms (SNPs) in C9orf72 on chromosome 9p21.2 (lowest p = 2.6 x 10(-12)) and 1 SNP in UNC13A on chromosome 19p13.11 (p = 1.0 x 10(-11)) as shared susceptibility loci for ALS and FTD-TDP. Conditioning on the 9p21.2 genotype increased statistical significance at UNC13A. A third signal, on chromosome 8q24.13 at the SPG8 locus coding for strumpellin (p = 3.91 x 10(-7)) was replicated in an independent cohort of 4,056 ALS patients and 3,958 controls (p = 0.026; combined analysis p = 1.01 x 10(-7)). Interpretation: We identified common genetic variants in C9orf72, but in addition in UNC13A that are shared between ALS and FTD. UNC13A provides a novel link between ALS and FTD-TDP, and identifies changes in neurotransmitter release and synaptic function as a converging mechanism in the pathogenesis of ALS and FTD-TDP.

The brain endocannabinoid system plays a crucial role in emotional processes. We have previously identified an important role for endocannabinoids in social play behavior, a highly rewarding form of social interaction in adolescent rats. Here, we tested the hypothesis that endocannabinoid modulation of social play behavior occurs in brain regions implicated in emotion and motivation. Social play increased levels of the endocannabinoid anandamide in the amygdala and nucleus accumbens (NAc), but not in prefrontal cortex or hippocampus of 4- to 5-week-old male Wistar rats. Furthermore, social play increased phosphorylation of CB1 cannabinoid receptors in the amygdala. Systemic administration of the anandamide hydrolysis inhibitor URB597 increased social play behavior, and augmented the associated elevation in anandamide levels in the amygdala, but not the NAc. Infusion of URB597 into the basolateral amygdala (BLA) increased social play behavior, and blockade of BLA CB1 cannabinoid receptors with the antagonist/inverse agonist SR141716A prevented the play-enhancing effects of systemic administration of URB597. Infusion of URB597 into the NAc also increased social play, but blockade of NAc CB1 cannabinoid receptors did not antagonize the play-enhancing effects of systemic URB597 treatment. Last, SR141716A did not affect social play after infusion into the core and shell subregions of the NAc, while it reduced social play when infused into the BLA. These data show that increased anandamide signaling in the amygdala and NAc augments social play, and identify the BLA as a prominent site of action for endocannabinoids to modulate the rewarding properties of social interactions in adolescent rats.

Item does not contain fulltext A large genome-wide screen in patients with sporadic amyotrophic lateral sclerosis (ALS) showed that the common variant rs12608932 in gene UNC13A was associated with disease susceptibility. UNC13A regulates the release of neurotransmitters, including glutamate. Genetic risk factors that, in addition, modify survival, provide promising therapeutic targets in ALS, a disease whose etiology remains largely elusive. We examined whether UNC13A was associated with survival of ALS patients in a cohort of 450 sporadic ALS patients and 524 unaffected controls from a population-based study of ALS in The Netherlands. Additionally, survival data were collected from individuals of Dutch, Belgian, or Swedish descent (1767 cases, 1817 controls) who had participated in a previously published genome-wide association study of ALS. We related survival to rs12608932 genotype. In both cohorts, the minor allele of rs12608932 in UNC13A was not only associated with susceptibility but also with shorter survival of ALS patients. Our results further corroborate the role of UNC13A in ALS pathogenesis. 01 maart 2012

Item does not contain fulltext Mutations in the valosin-containing protein (VCP) gene were recently reported to be the cause of 1%-2% of familial amyotrophic lateral sclerosis (ALS) cases. VCP mutations are known to cause inclusion body myopathy (IBM) with Paget's disease (PDB) and frontotemporal dementia (FTD). The presence of VCP mutations in patients with sporadic ALS, sporadic ALS-FTD, and progressive muscular atrophy (PMA), a known clinical mimic of inclusion body myopathy, is not known. To determine the identity and frequency of VCP mutations we screened a cohort of 93 familial ALS, 754 sporadic ALS, 58 sporadic ALS-FTD, and 264 progressive muscular atrophy patients for mutations in the VCP gene. Two nonsynonymous mutations were detected; 1 known mutation (p.R159H) in a patient with familial ALS with several family members suffering from FTD, and 1 mutation (p.I114V) in a patient with sporadic ALS. Conservation analysis and protein prediction software indicate the p.I114V mutation to be a rare benign polymorphism. VCP mutations are a rare cause of familial ALS. The role of VCP mutations in sporadic ALS, if present, appears limited. 01 april 2012

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 x 10(-4) in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.30 x 10(-9). This SNP showed robust replication in the second cohort (P = 1.86 x 10(-6)), and a combined analysis over the two stages yielded P = 2.53 x 10(-14). The rs12608932 SNP is located at 19p13.3 and maps to a haplotype block within the boundaries of UNC13A, which regulates the release of neurotransmitters such as glutamate at neuromuscular synapses. Follow-up of additional SNPs showed genome-wide significance for two further SNPs (rs2814707, with P = 7.45 x 10(-9), and rs3849942, with P = 1.01 x 10(-8)) in the combined analysis of both stages. These SNPs are located at chromosome 9p21.2, in a linkage region for familial ALS with frontotemporal dementia found previously in several large pedigrees.

The interaction of a myriad of molecular factors is necessary for proper development of mDA circuitry. Although significant progress has been made in uncovering the mechanisms involved in mDA neuron development, our understanding of the role played by specific classes of molecules (such as non-coding RNAs) is still incomplete. To extend existing knowledge, we examined the cellular aspects of mDA neuron development (chapter 1). Using this information, we identified relevant timepoints for a comprehensive transcriptomic profiling study of mouse mDA neurons and employed bioinformatics analysis to uncover patterns of lncRNA and circRNA expression during embryonic and postnatal development (chapter 1). We selected specific circRNA candidates for further functional investigation (chapter 2). Through in vitro and in vivo knockdown of a particular circRNA, we discovered several cellular and molecular changes in normal mDA neuron development. Advances in sequencing techniques have facilitated the assessment of molecular profiles at the single-cell level in a more efficient and rapid manner. However, the definition of subtypes remains a subject of debate. Therefore, to gain a better understanding of the extent of mDA system heterogeneity, it is crucial to study cellular properties, such as morphology and electrophysiology. Consequently, we utilized intersectional mouse genetics to investigate different subsets of mDA neurons and their individual morphology (chapter 3). The mDA system can be broadly categorized into the VTA and the SNc. These two regions receive and respond to distinct molecular signals that guide the neurons to their final positions after birth. In chapter 4, we made a novel discovery regarding a long-range mechanism that promotes cellular migration. Furthermore, we demonstrate the importance of the localization of other cell types (specifically, GABAergic neurons) for the proper placement of mDA neurons.

Skeletal muscles are highly resilient due to the capacity to regenerate damaged muscle tissue. Despite the regenerative capacity, pathological conditions can lead to reduced muscle mass and strength, also known as muscle wasting. Muscle wasting, seen in pathological conditions, is often progressive and concomitant with excessive or prolonged inflammation, fibrosis and fat infiltration. Aging is a risk factor for a large number of diseases that can result in muscle wasting. The prevalence of sarcopenia, an age-associated decline in skeletal muscle mass and strength in elderly, surges as life expectancy increases. In combination with muscle wasting caused by neuromuscular disorders, the burden of muscle wasting on society is significant. Gaining a better understanding of the cellular and molecular processes underlying muscle wasting conditions is critical for creating better treatments. Aging and thereby age-associated muscle wasting are multifactorial involving changes in different tissues and multiple cell systems. This thesis demonstrates how different omics approaches, like transcriptomics, proteomics and metabolomics, can elucidate the complexity of those molecular mechanisms and cellular processes involved in muscle wasting conditions. Employing a combination of different omics-techniques (multi-omics) specifically proved powerful and will continue to illuminate impaired mechanisms and consequently help to battle muscle wasting.

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease that affects motor neurons in the cerebral cortex, brain stem and spinal cord. Several genetic mutations have been identified that cause or increase the risk of developing ALS. Among them, repeat expansions in intronic or exonic regions of several genes have been linked to ALS, including in C9ORF72 and Ataxin-2 (ATXN2) genes. Expanded repeats in either case lead to cytotoxicity by protein loss-of-function as well as toxic gain-of-function of the repeat. The GGGGCC hexanucleotide repeat expansion (HRE) in C9orf72 is the most common cause of ALS identified to date. The HRE results in C9orf72 haploinsufficiency, HRE RNA foci, and dipeptide repeats resulting from repeat-associated non-ATG translation of the HRE. In ATXN2 protein, intermediate expansion of the polyQ stretch is a risk factor for developing ALS. In this thesis, we approach different aspects of C9ORF72 and ATXN2 repeat expansions in ALS mainly using induced pluripotent stem cell (iPSC) -derived models. In chapter 2 we review the use of central nervous system organoid models in neurodegeneration. We discuss limitations and future perspectives and ultimately provide a framework for the development of organoid-based organ-on-a-chip models for motor neuron disease. In chapter 3 we describe a cerebral organoid model where microglia innately develop. Organoid-derived microglia are similar to adult post-mortem microglia in morphology, transcriptional profile, and inflammatory response. These microglia are competent in phagocytosis and overall represent a valuable model for studying the complex interplay of different brain cell populations in development and disease. In chapter 4 we use the model developed in chapter 3 to study C9orf72 hexanucleotide HRE-associated brain pathology. We show that C9ORF72 HRE makes embryoid bodies significantly smaller than controls. Additionally, we find that at a mature stage, C9orf72 ALS organoids have altered expression of neural progenitor and forebrain markers, hinting at abnormal organoid development. At a later timepoint, C9ALS organoids are smaller than controls which could be a reflection of ALS-associated neural atrophy. In chapter 5, we combine mouse and patient iPSC-derived models to study the contribution of the Ataxin-2 polyQ intermediate repeat expansion in ALS. In both models, transcriptomic signatures show altered expression of genes involved in lipid metabolism, known to be associated with ATXN2 polyQ. We show that ATXN2 intermediate repeat expansion is sufficient to induce both loss- and toxic gain-of-function phenotypes which impact lipid metabolism. Finally, in chapter 6 we discuss technical aspects of iPSC models and of the origin of microglia in cerebral organoids. We then discuss a broader role of lipid metabolism in ALS. Lastly, we highlight the contribution, synergy and overlap between Ataxin-2 and C9orf72. The findings described in this thesis will hopefully contribute to the understanding of repeat expansions in ALS.

During the development of the brain, different types of neuronal and non-neuronal cell types are formed. These cells migrate through the developing brain to arrive at their final location. In addition, they send out axons in order to establish connections with other brain areas. These processes are controlled and guided by axon guidance proteins. This ultimately forms a fully functioning brain that performs complex functions. Studying the RNA profile of individual cells provides insight into the heterogeneity, and therefore complexity, of the brain. This can be done with the aid of 'single-cell RNA sequencing'. With this technique, the RNA profile of individual cells is mapped. With this information, the properties of subgroups of cells can be investigated. An example of a heterogeneous brain region is the habenula. This structure is present in both humans and mice. Research into the function of the habenula has distinguished between two subgroups within the habenula: the medial and lateral habenula. Recently, however, it has been shown that there are more than two subgroups within the habenula. How these subgroups arise during brain development, however, remained unknown. In chapter 2, we characterize mouse habenula cell types one day before birth, on embryonic day 18, using single-cell RNA sequencing. We find that at the end of embryogenesis multiple subgroups of habenula neurons are already present. In addition, our experiments show that on day 18 actively dividing cells are still present in the habenula. We also describe in detail where different cell types are located in the habenula. In chapter 3 we investigate the development of habenula neurons during embryogenesis and just after birth in mice. We analyze the RNA profile of developing habenula neurons at five embryonic timepoints and three postnatal timepoints. This allowed us to map how habenula neurons develop and mature during development. In Chapter 4 we discussed the technical aspects of analyzing data obtained in a single-cell RNA sequencing experiment. Given the high dimensionality of the data, the analysis can be complex. The complexity is increased when one wants to merge data obtained in different labs or from different experiments. However, such a comparison can be of enormous added value as it allows putting newly acquired data into context of previously acquired data. We looked specifically at how to deal with these challenges. A large part of the solution lies in removing batch effects. We tested the effectiveness of different methods that correct for batch effects. In chapter 5, we investigated the dynamics between axon guidance receptor Neogenin and two of its ligands: the signal proteins RGM and Netrin-1. These three molecules regulate important processes during development and in disease. We found that instead of competing for binding, the two ligands can simultaneously bind the receptor. This has important biological implications because during development and during disease these three molecules are present in overlapping locations in the brain. Our experiments show that the supercomplex suppresses the normal effects that binding of RGM alone or Netrin1 to the receptor Neogenin brings about.

Nearly 98 - 99% of the genome has little to no coding function and it is transcribed into non-coding RNAs (ncRNAs). These influence transcription, RNA processing and translation and include different sorts of molecules such as transfer RNAs (tRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs). RNA metabolism is extremely relevant in the brain, as RNA is dynamically expressed and shows highly regulated temporal and spatial patterns, including tissue- and cell-type specificity. Research has shown that ncRNAs participate in several processes including brain development, synaptic plasticity, cell death, oxidative stress and epigenetic modifications. Importantly, their expression is often perturbed in tissues affected by neurological disorders (e.g. Alzheimer’s disease, Autism spectrum disorder, Huntington's disease, glioma) and accounts for an important layer of complexity when trying to dissect brain disease-mechanisms. Epilepsy is considered the most common chronic brain disease by affecting more than 50 million people globally. Currently, it is considered as a complex disorder likely resulting from the interplay between intrinsic (genetic) and extrinsic factors such as head trauma, infection episodes or brain tumours. Mesial temporal lobe epilepsy (mTLE) is a common form of focal epilepsy in which seizures originate from the amygdalo-hippocampal regions. An initial injury event (status epilepticus) is thought to trigger brain alterations (epileptogenesis, or ‘latent phase’) which will later develop into spontaneous seizures and, in some cases, hippocampal sclerosis. The initial treatment for the majority of epilepsy patients is based on antiepileptic drugs (AEDs). However, more than 30% of epilepsy patients continue to experience seizures despite AED treatment. This condition, known as ‘refractory epilepsy’, is present in 80% of mTLE patients. As a result, there is an urgent unmet need for innovative therapeutic approaches for treating refractory epilepsy. The pathological mechanisms leading to temporal lobe epilepsy (TLE) are not fully comprehended. The extensive use of animal models along with observations from human tissue have underlined important features. Common deregulated mechanisms include: neuronal loss, granule cell mossy fiber sprouting, activated astrocytes and microglia, decreased glutamine synthetase, down-regulation of ion channels and neurotransmitters, altered synaptic plasticity and dysfunction of the blood-brain barrier. These processes co-exist in the epileptic brain and ultimately lead to excitotoxicity and neuronal hyperexcitability. How altered ncRNA expression influences these processes remains elusive. In this dissertation, we aimed to explore the profile and function of the non-coding transcriptome in the healthy and diseased brain. Individually, the chapters in this dissertation illustrate how different ncRNA classes may contribute to disease-related mechanisms. Together, they provide a framework to rethink novel therapeutic approaches for brain disease which remain untreatable or associated with poor treatment outcomes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons., Dutch Research Council (NWO); VENI Scheme Grant; VIDI Grant; Prinses Beatrix Spierfonds; Neuromuscular Fellowship Grant; Medical Research Council (MRC); Clinical Infrastructure Award; Epidemiology Unit; Integrative Epidemiology Unit; Canadian Institutes of Health Research; IWT; National Institute on Aging; National Health and Medical Research Council (NHMRC); Enabling Grant; NHMRC/Australian Research Council Strategic Award; NHMRC; NHMRC Centre of Research Excellence Grant; National Health and Medical Research Council of Australia (NHMRC) Research Fellowship; United Kingdom, Medical Research Council; Economic and Social Research Council; European Union (EU); Horizon 2020; European Community's Health Seventh Framework Programme; EuroMOTOR; European Research Council (ERC); Research and Innovation Programme; EScORIAL; ALS Foundation Netherlands; Alzheimer’s Society PhD Studentship; ARSla Funding; Biogen; University of Bristol; Motor Neurone Disease Association (MNDA); NIHR Maudsley Biomedical Research Centre; Dutch Ministry of Education, Culture, and Science; Netherlands Organization for Scientific Research (NWO; BRAINSCAPES); Gravitation Program; ALS Liga België; National Lottery of Belgium; KU Leuven Opening the Future Fund; KU Leuven Funds, “Een Hart voor ALS”, “Laeversfonds voor ALS Onderzoek” and the “Valéry Perrier Race against ALS Fund”; E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders; ALS Liga België; “Live now” Charity Foundation; Moscow ALS palliative Care Service; Canadian Institutes of Health; Research Australia; Ice Bucket Challenge Grant; NIH Intramural Research Programs; FightMND Mid-Career Fellowship; NIHR Senior Investigator; Sheffield NIHR Biomedical Research Centre; Motor Neurone Disease Association; National Institute for Health Research (NIHR) Biomedical Research Centre; Maudsley NHS Foundation Trust; King’s College London; NIHR Senior Investigator Award; Netherlands Organization for Health Research and Development; Vici Scheme; Netherlands Organization for Health Research and Development STRENGTH Project; PPP Allowance

The brain is a complex and versatile organ that undergoes various phases of development. This thesis focuses on the moment when neurons migrate from their birthplace to different brain areas and start to make connections with other cells to form neural circuits. During this phase, axon guidance molecules play an important role in establishing proper neural organization and connectivity. The principle of axon guidance is that growing axons and migrating cells rely on intermediate targets and guidance cues presented over short or long distances along their paths in order to establish neuronal connections. Growing axons extend their growth cones to scan the environment and respond to axon guidance cues that provide direction towards or away from specific regions. Semaphorins are axon guidance molecules that are well-known for their function as attractants or repellents. Although only a limited set of semaphorins has been identified, accumulating evidence indicates that distinct molecular mechanisms act to diversify the effects of semaphorins, making semaphorins multifunctional molecules that control a wide variety of cellular events. Chapter 2 comprises an extended introduction and description of general features and novel aspects of semaphorin function and signaling, including the role of their receptors; plexins and neuropilins. The focus is on the contribution of these molecules to the regulation of cell migration and the formation of neuronal connections. Semaphorin6A (Sema6A) is an example of a member of the semaphorin protein family that plays a key role in the nervous system, yet we know very little about its multifunctional role and diverse signaling mechanisms. In this thesis, the aim is to determine and specify these Sema6A signaling events during the development of the brain. Throughout the subsequent chapters of the thesis, three topics will be addressed: 1. Sema6A reverse signaling in vivo (Chapters 3 and 4) 2. Sema6A cis interaction and the specific structural domains involved in this interaction (Chapter 5) and 3. Novel functions of Sema6A in the diseased brain (Chapter 6). This thesis provides an overview of recent developments in the field of axon guidance research focusing on semaphorins, including the role of their interactors; plexins and neuropilins (Chapter 2). Furthermore, it focuses on Sema6A, which is known to play important roles during the development of the nervous system. The overall aim of this thesis is to study the multifunctional character of Sema6A by selectively exploring functions of the intracellular domain (Chapter 3) and reverse signaling events (Chapter 4), interactors and regulators involved in Sema6A signaling pathways (Chapter 4), cis interaction domains (Chapter 5) and to specify novel roles during puberty onset and neuronal disease (Chapter 6). These previously unexplored functions help to understand the complexity of semaphorin signaling pathways and provide an extensive and in-depth overview of Sema6A signaling during brain development. The findings described in this thesis contribute to the knowledge of complex and dynamic cellular and molecular events that help to understand brain development and disorders.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade., The LifeTime initiative is an ambitious, multidisciplinary programme that aims to improve healthcare by tracking individual human cells during disease processes and responses to treatment in order to develop and implement cell-based interceptive medicine in Europe.

This thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and regulation of the transgene expression were also investigated.Due to the permanent nature of gene therapy, a method to modulate the transgeneexpression is desirable.

During embryonic development neurons migrate and grow their protrusions (neurites) over long distances in a strictly orchestrated manner to form complex neuronal networks. Subtle changes in neuronal network formation may lead to various neurological disorders ranging from congenital mirror movements, to autism spectrum disorders and Parkinson’s disease. Apart from canonical axon guidance proteins, cell adhesion molecules (CAMs) are indispensable for proper neuron migration and the formation of correct synaptic connections. Interestingly, the expression and function of these proteins is spatiotemporally regulated, resulting in the potential to control a disproportionally large number of guidance decisions. In this thesis some of these regulatory strategies, including post-transcriptional regulation by microRNAs (miRNAs) and membrane targeting of CAMs by Molecules interacting with Casl (MICALs), are highlighted, and combined with a detailed description of dopamine axon bundle development and a review on the role of canonical axon guidance proteins in nervous system diseases. MiRNAs are small, non-coding RNA molecules that generally downregulate translation of their target mRNAs. In the human brain, over thousand miRNAs have been identified, however functional roles are unknown for most of them. In this thesis, two different strategies were used to clarify roles of miRNAs during axon development. First, a profiling study identified groups of miRNAs that are involved in specific neurodevelopmental stages. Second, a high-throughput whole-miRNA-nome cellomics screen lead to the identification of the miRNA-135-family as regulators of neurite outgrowth by targeting Krüppel-like factor 4 (KLF4). This provides interesting new opportunities for axon regeneration therapy development, since it is long known that KLF4 is an important downregulator of axon growth after injury. In many brain nuclei, including the hippocampus, synaptic connections are organized in specific laminae. CAMs play an important role directing the formation of these lamina-specific synapses, but the mechanisms behind these processes are still unclear. We discovered that the redox protein MICAL-1 is involved in membrane-targeting of specific CAMs (NCAM and CHL1) by regulating actin cytoskeleton rearrangements in the growth cone. By that, it controls lamina-specific targeting of dentate gyrus mossy fiber synapses in the hippocampus in two ways: 1) it regulates the fasciculation of mossy fibers and confines them to the supra- and infrapyramidal axon bundles, and 2) it regulates subcellular-specific synapse formation of dentate gyrus mossy fibers with proximal dendrites of CA3 pyramidal neurons and restricts synaptic formation to the stratum oriens and the stratum lucidum of the hippocampus. Using a novel genetic mouse model called ITC, we were able to distinguish developing midbrain dopaminergic neurons and axons from the substantia nigra compacta (SNc) by fluorescent labeling. We performed a detailed analysis of their specific migration-, axon growth- and striatal innervation patterns from early on in embryonic development, and discovered that SNc axons are segregated dorsally in the medial forebrain bundle and project directly in a dorsal-to-dorsal fashion to the striatum. This suggests a specific axon guidance mechanism, which we will be able to investigate further using these novel transgenic mice. Further, these mice provide the opportunity investigating pre-symptomatic alterations in dopaminergic connectivity in Parkinson’s disease.

Disruption in the genes coding for the neural cell adhesion molecules contactin-4 (Cntn4), contactin-5 (Cntn5), and contactin-6 (Cntn6) contribute to an increased risk of neuropsychiatric disorders, particularly autism spectrum disorders (ASD). This indicates that the absence or dysfunction of these proteins may disrupt normal brain development. While Cntn4, Cntn5, and Cntn6 are expressed in distinct but overlapping brain regions in rodents, the gross neuroanatomy of the respective null-mutant mice is not affected. More detailed analyses of specific processes in neurodevelopment did demonstrate impairments in several neurobiological processes, including neurite outgrowth, synaptogenesis, neural survival, guiding projections and terminal branches of axons in forming neural circuits. Although studies so far indicate distinct neuroanatomical expression patterns and behavioral phenotypes in the respective null-mutant mice, very little is still known about the molecular mechanisms underlying the biological pathways in which these Cntns participate. The common structural architecture of the Cntn subgroup indicates the necessity of binding partners in order for them to facilitate any neurobiological functions. While Cntns are membrane-linked, but not membrane-spanning, the involved mechanisms of action must rely on a signalling function of the Cntn protein itself or the interaction with other membrane-spanning proteins. Abundant evidence for contactin-1 (Cntn1) and contactin-2 (Cntn2) show that cis- and trans-interaction with several membrane-spanning proteins is part of their repertoire. Therefore, the aim of this thesis is to unravel the neurobiological mechanisms of Cntn4, Cntn5, and Cntn6, with emphasis on Cntn6. Through proteomics novel and known interacting proteins were identified for these three contactins. The interaction of Cntn6 with the cell adhesion G protein-coupled receptor latrophilin-1 (Lphn1, aka CIRL, ADGRL1) was investigated in detail, showing that Cntn6 inhibited Lphn1-mediated apoptosis. These results suggest that Lphn1 functions as a dependence receptor, which induces neuronal apoptosis only in absence of Cntn6. Indeed, neuroanatomical analysis of the visual cortex of Cntn6 null-mutantmice displayed an increase in apoptosis, demonstrating the significant consequences in vivo. Furthermore, it provides a novel look into modes of action of Cntns and into the consequences for brain development. The identification of cell adhesion properties of Cntn6 in the cerebral cortex points to a broader function for Cntn6 in neurodevelopment than was previously shown. This was further emphasized by the ability of Cntn6 to interact with multiple membrane-spanning proteins with each having several interactions of their own, including the well-known and ASD-implicated Nrxn1-Nlgn1 pathway. This may represent the leading edge for integrated protein networks important for brain development, maturation and plasticity. It may provide valuable starting points for future studies to unravel how absence of Cntns impair neurobiological processes, and ultimately lead to ASD pathophysiology.

The development of a fully functional brain requires the orchestration of simultaneously coordinated migration, projection and network formation to connect millions of neurons. When neuronal connections are lost or neurons die as a result of neurodegenerative disease or injury, the regenerative capacities of the adult central nervous system (CNS) are limited. In order to develop clinical strategies to improve this regenerative potential, we need to 1) increase our understanding of the guidance cues and cellular processes that regulate neural circuit formation during development, and 2) investigate the pathological changes that occur following adult CNS injury and/or disease. These insights will lead to novel therapeutic targets for future (pre)clinical studies. This thesis contributes to our understanding of; 1) signaling downstream of the axon guidance receptor Neogenin; 2) intrinsic and extrinsic regulation of neuronal migration; and 3) molecular changes associated with human epilepsy. These approaches not only identify novel factors involved in the regulation of neural development, but also characterize the pathology of a neurological disease.