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Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.

Disease modeling for neurodevelopmental disorders has long been a challenging endeavor, necessitating innovative approaches to understand the underlying mechanisms. Traditional animal models, while informative, often fail to fully capture the intricacies of human brain development and associated diseases. FOXG1 syndrome, caused by mutations in the Forkhead box protein G1 (FOXG1) gene and characterized by forebrain developmental abnormalities, represents one such disorder with a range of neurological symptoms including motor deficits, intellectual retardation, and seizures. In recent years, cortical brain organoid models derived from human pluripotent stem cells have emerged as a revolutionary tool in neuroscience research, providing a three-dimensional representation of the human brain. These organoids exhibit structural and functional resemblance to developing brains, encompassing the formation of diverse cell types and the establishment of functional networks. In this study, we were successfully able to develop a cortical organoid model using both healthy and patient derived cell lines for investigating FOXG1 syndrome, with the patient lines accurately reproducing the microcephaly phenotype observed in FOXG1 syndrome We explored strategies to enhance the cortical identity of the model using Wingless/Integrated pathway (WNT pathway) inhibition, achieving promising results. Furthermore, we successfully recapitulated neural tube development by constructing a single rosette organoid, thereby increasing its biomimetic properties. Our findings highlight the potential of cortical organoids as powerful tools for studying human neurodevelopment and neurodevelopmental disorders, offering new avenues for understanding disease pathogenesis and facilitating the development of targeted therapeutic interventions.

The vertebrate eye anlage grows out of the brain and folds into bilayered optic cups. The eye is patterned along multiple axes, precisely controlled by genetic programs, to delineate neural retina, pigment epithelium, and optic stalk tissues. Pax genes encode developmental regulators of key morphogenetic events, with Pax2 being essential for interpreting inductive signals, including in the eye. PAX2 mutations cause ocular coloboma, when the ventral optic fissure fails to close. Previous studies established that Pax2 is necessary for fissure closure and to maintain the neural retina -- glial optic stalk boundary. Using a Pax2GFP/+ knock-in allele we discovered that the mutant optic nerve head (ONH) lacks molecular boundaries with the retina and RPE, rendering the ONH larger than normal. This was preceded by ventronasal cup mispatterning, a burst of overproliferation and followed by optic cup apoptosis. Our findings support the hypothesis that ONH cells are tripotential, requiring Pax2 to remain committed to glial fates. This work extends current models of ocular development, contributes to broader understanding of tissue boundary formation and informs the underlying mechanisms of human coloboma.

The self-renewal activity of neural stem cells (NSCs) has been suggested to decrease with aging, resulting in age-dependent declines in brain function, such as presbyopia and memory loss. The molecular mechanisms underlying decreases in NSC proliferation with age need to be elucidated in more detail to develop treatments that promote brain function. We have previously reported that the expression of esophageal cancer-related gene 4 (Ecrg4) was upregulated in aged NSCs, whereas its overexpression decreased NSC proliferation, suggesting a functional relationship between Ecrg4 and NSC aging. Using Ecrg4-deficient mice in which the Ecrg4 locus was replaced with the lacZ gene, we here show that Ecrg4 deficiency recovered the age-dependent decline in NSC proliferation and enhanced spatial learning and memory in the Morris water-maze paradigm. We demonstrate that the proliferation of Ecrg4-deficient NSCs was partly maintained by the increased expression of Foxg1. Collectively, these results determine Ecrg4 as a NSC aging factor.

The differential diagnostics in Rett syndrome has evolved with the development of next generation sequencing-based techniques and many patients have been diagnosed with other syndromes or variants in newly described genes where the associated phenotype(s) is yet to be fully explored. The term Rett-like refers to phenotypes with distinct overlapping features of Rett syndrome where the clinical criteria are not completely fulfilled. In this study we have combined a review of Rett-like disorders with data from a Danish cohort of 35 patients with Rett-like phenotypes emphasizing the diagnostic overlap with Pitt-Hopkins syndrome, Cornelia de Lange syndrome with SMC1A variants, and epileptic encephalopathies, for example, due to STXBP1 variants. We also found a patient with a pathogenic variant in KCNB1, which has not been previously linked to a Rett-like phenotype. This study underlines the clinical and genetic heterogeneity of a Rett syndrome spectrum, and provides an overview of the Rett syndrome-related genes described to date, and hence serves as a guide for diagnosing patients with Rett-like phenotypes.

The cortex consists of 100s of neuronal subtypes that are organized into distinct functional regions; however, the mechanisms underlying cell fate determination remain unclear. Foxg1 is involved in several developmental processes, including telencephalic patterning, cell proliferation and cell fate determination. Constitutive disruption of Foxg1 leads to the transformation of cortical neurons into Cajal-Retzius (CR) cells, accompanied by a substantial expansion of the cortical hem through the consumption of the cortex. However, rather than the induction of a cell fate switch, another group has reported a large lateral to medial repatterning of the developing telencephalon as the explanation for this change in cell type output. Here, we conditionally disrupted Foxg1 in telencephalic progenitor cells by crossing Foxg1fl/fl mice with Nestin-CreERTM mice combined with tamoxifen (TM) induction at distinct developmental stages beginning at E10.5 to further elucidate the role of FoxG1 in cell fate determination after telencephalon pattern formation. The number of dentate gyrus (DG) granule-like cells was significantly increased in the cortex. The increase was even detected after deletion at E14.5. In vivo mosaic deletion and in vitro cell culture further revealed a cell-autonomous role for FoxG1 in repressing granule cell fate. However, the cortical hem, which is required for the patterning and the development of the hippocampus, was only slightly enlarged and thus may not contribute to the cell fate switch. Lef1 expression was significantly upregulated in the lateral, cortical VZ and FoxG1 may function upstream of Wnt signaling. Our results provide new insights into the functions of FoxG1 and the mechanisms of cell fate determination during telencephalic development.

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the degeneration and death of both upper and lower motor neurons for which there is no cure currently available. The degeneration of motor neurons leads to the inability to carry out voluntary movement and death usually occurs 3-5 years after disease onset by respiratory failure. The most common cause of familial ALS is associated with mutations in the superoxide dismutase gene (SOD1). The SOD1 gene codes for an enzyme that is ubiquitously expressed and is involved in neutralizing reactive oxygen species. Mutations in the SOD1 gene lead to a toxic gain-of-function and astrocytes with mutated SOD1 has been shown to kill motor neurons by releasing toxic factors. In this project the gene-editing tool CRISPR/Cas9 was used to knock down SOD1 in forebrain neural progenitor cells (FB NPCs) using single guide RNAs (sgRNAs) to target the SOD1 gene specifically. The embryonic stem cell line RC17 and the induced pluripotent and patient-specific stem cell line SOD1L144P were differentiated to forebrain neural progenitor cells (FB NPCs) using a 16-day protocol. Lentivirus vectors were used to deliver the Cas9 protein, a sgRNA targeting either exon 2 (sgRNA:E2) or exon 3 (sgRNA:E3) and the green-fluorescent protein (GFP) as a reporter gene into the cells. The success of the differentiation was validated based on immunocytochemistry staining for the expression of FOXG1 and nestin, and the transduction on the presence of GFP. Staining for SOD1 was carried out to study the SOD1 knock-down on a protein level. The knock-down of SOD1 was also analysed based on mRNA expression using qPCR with a SOD1-specific primer. The results from the qPCR showed that the cells transduced with sgRNA:E2 and sgRNA:E3 had a reduced expression of SOD1 in both cell lines. In the case of sgRNA:E3 the reduction was more than four-fold in both cell lines., Under projektets gång användes gen-editeringsmetoden CRISPR/Cas9 för att förhindra uttrycket av en gen som är associerad med en familjär form av ALS i två stamcellslinjer som hade blivit differentierade till hjärnceller. Genom genuttrycksanalys visades att CRISPR/Cas9 hade lyckats orsaka en fyrfaldig minskning av SOD1-uttryck i båda cellinjer. Amyotrofisk lateralskleros, förkortat ALS är en neuromuskulär sjukdom som kännetecknas av degenerationen av motor neuroner, vilka är nödvändiga för att utföra kontrollerbara rörelser. De primära symptomen för ALS är muskelatrofi, muskelsvaghet, muskelkramp samt tal-och sväljsvårigheter, och inträffar oftast mellan åldrarna 55 och 75 år. Det finns inget botemedel mot ALS och död inträffar vanligtvis 3–5 år efter de första symptomen genom andningssvikt. Ungefär 3 personer per 100 000 per år utvecklar ALS och orsaken kan både vara familjär (genetisk) eller sporadisk. Cirka 10% av alla fall är familjära där patienterna har en genetisk predisposition att utveckla sjukdomen. De vanligaste fallen av ALS är associerade med mutationer i superoxide dismutase (SOD1) genen. Även om den exakta mekanismen bakom patologin är inte fullt förstådd så tyder mycket på att det bildas proteinaggregat av SOD1 som är toxiska för cellerna. Många försök har gjorts att knocka ner uttrycket av SOD1, men dessvärre har resultaten inte varit kliniskt signifikanta. Dessutom har dessa försök inte lett till permanenta förändringar i genen utan endast minskat uttrycket av SOD1 för stunden. Genom att använda gen-editeringsmetoden CRISPR/Cas9 kan ändringar i SOD1-genen göras på en DNA-nivå vilket skulle leda till permanenta förändringar i arvsmassan. CRISPR/Cas9 består av två komponenter; ett protein som kallas Cas9 och är ett enzym som kan klippa i DNA molekyler and en guide-molekyl som leder Cas9 till rätt plats i genomet där det sen klipper. När en cell försöker reparera skadan från Cas9 introducerar reparationssystemet fel i genen, vilket leder till att den i

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K+ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf +/− mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.

Human epidermal growth factor receptor 2 (HER2) overexpressing breast cancers (HER2+ breast cancer) are associated with an aggressive disease course. This thesis is focused on improving the understanding of the biological signature of HER2+ breast cancer. In Paper I, we identified a common deletion spanning the SLC25A43 gene which codes for a mitochondrial transport protein. Loss of heterozygosity in this gene was confirmed in an extended cohort of HER2+ breast cancer and in other types of cancers. Protein expression analysis of SLC25A43using immunohistochemistry (IHC) in HER2+ breast cancers showed that tumours with negative or low expression of SLC25A43 had lower S-phase fraction compared to tumours with medium or high expression, indicating its possible role in cell proliferation. Absence of mutations in this gene in HER2+ breast cancers led to Paper II where DNA methylation in the SLC25A43 gene was interrogated using Pyrosequencing. HER2+ breast cancer with no deletion in the SLC25A43 gene showed higher methylation in the CpG island (CGI), suggesting methylation in the CGI as an alternate mechanism for SLC25A43 gene inactivation. Methylation in the CGI and in the adjacent shores of the SLC25A43 gene was associated with negative oestrogen receptor status and positive lymph node status. In Paper III, genome-wide DNA methylation analysis of HER2+ breast cancer and normal breast tissue revealed hypermethylation in HER2+ breast cancer affecting particularly the homeobox gene family when compared to normal. We identified a total of 73 candidate genes showing differential methylation in HER2+ breast cancer and external validation of gene expression in a selected group of these genes revealed lowered mean expression in HER2+ breast cancer, warranting future clinical studies of these candidate genes. In Paper IV, we investigated expression and localisation of phosphorylated (p) Akt and FOXO3a and FOXG1 in HER2+ breast cancer using IHC. Cytoplasmic expression of pFOXO3a w

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.

Introduction: Breast cancers with human epidermal growth factor receptor (HER) 2 gene amplification or protein overexpression (HER2+ breast cancer) is associated with poor prognosis. Activated HER2 receptors dimerise and activate the oncokinase Akt; which phosphorylate the tumour suppressor protein Forkhead box O3a (FOXO3a), repressing its transcriptional activity. Oncogenic FOXG1 can act as a transcriptional repressor for genes which are transcriptionally activated by FOXOs and phosphorylation of FOXG1 via Akt causes its nuclear export in differentiated cells. To better understand the AKT/FOXO3a/FOXG1 connection and their clinical relevance, we investigated the expression and localisation of pAkt, pFOXO3a and FOXG1 in HER2+ breast cancer. Methods: Immunohistochemical analysis was performed to determine the expression and localisation of pAkt, pFOXO3a and FOXG1 on tissue microarray constructs from HER2+ primary breast tumours (n=91) and their relation to clinic pathological parameters were analysed. Results: Nuclear expression of pAkt was found to be correlated to nuclear (p<0.001) as well as cytoplasmic expression of pFOXO3a (p = 0.006), while cytoplasmic expression of pAkt was found to be correlated to cytoplasmic expression of pFOXO3a (p<0.001). Nuclear expression of pFOXO3a was inversely correlated to cytoplasmic expression of FOXG1 (p=0.003). Cytoplasmic expression of pFOXO3a was found to be associated with sentinel node metastasis (p=0.011), while cytoplasmic FOXG1 expression was correlated to negative progesterone receptor status (p=0.008). Conclusion: Our results suggest that the expression and localisation of pAkt and pFOXO3a is interconnected, while the expression of FOXG1 is connected to pFOXO3a. Our findings indicate the biological value of expression as well as localisation of these proteins in HER2+ breast cancer.