Tesis Doctorals - Departament - Biomedicina

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Novel molecular mechanisms contributing to cognitive impairment in schizophrenia
(Universitat de Barcelona, 2025-07-11) Galán Ganga, Marcos; Giralt Torroella, Albert; Universitat de Barcelona. Departament de Biomedicina
[eng] Schizophrenia is a neuropsychiatric syndrome that affects around 1% of the world population (Marder & Cannon, 2019). Its age of onset typically occurs within the second and third decades of life (Velligan & Rao, 2023). It is characterized by the presence of psychosis (e.g. hallucinations, delusions and disorganized speech), negative symptoms (e.g. social withdrawal, anhedonia and apathy), and cognitive deficits (e.g. social cognition deficits, impaired executive functions and working memory) (Marder & Cannon, 2019). Antipsychotic treatments can help to manage positive symptoms during acute episodes. However, their side effects and limited benefits on sociability and cognition often result in poor medication adherence and increased risk of relapse. Consequently, there is a significant unmet medical need, particularly for treatments targeting those cognitive symptoms that emerge in the premorbid phase and affect nearly 98% of patients (Harvey et al., 2022; Mihaljević-Peleš et al., 2019). The complexity of the disease relies not only on its wide variety of symptoms, but also from the different heritable and environmental risk factors that influence the development of schizophrenia (Marder & Cannon, 2019; McCutcheon et al., 2020; Jauhar et al., 2022). Research made over the last decades has identified several molecular pathways impaired in the disorder, with neurochemical disturbances related to the dopaminergic and glutamatergic systems having a prominent role in its pathophysiology (Jauhar et al., 2022). Recent evidence has revealed that impaired GABAergic and serotoninergic neurotransmission, neurodevelopmental abnormalities and inflammation could also play a role in the onset and progression of schizophrenia (De Jonge et al., 2017; Gaitonde et al., 2024; Marder & Cannon, 2019; Jauhar et al., 2022; E. E. Lee et al., 2017; Hong & Bang, 2020). Although anatomical and functional dysfunctions have been found in different brain areas from diseased patients, the hippocampus has been suggested to play a pivotal role. Different morphological, electrophysiological, synaptic and molecular impairments affecting this brain region have been associated to schizophrenia and in close relation with to some of its positive, negative and cognitive symptoms (Harrison, 2004; Wegrzyn et al., 2022). Notwithstanding the progresses made to enhance our comprehension of this illness, there remains a need for novel approaches and improved translational models that may recapitulate more accurately its underlying pathological mechanisms. Latest reports have found altered levels of small RNAs (sRNAs) in post-mortem brain samples of patients affected by schizophrenia and other neuropsychiatric disorders (Yoshino & Dwivedi, 2020). sRNAs are non-coding RNAs with less than 200 nucleotides that do not code for proteins but play an essential role regulating messenger RNA (mRNA) expression, stability and translation, with an important function in health and disease (Yoshino & Dwivedi, 2020). Among the different sRNA biotypes, microRNAs (miRNAs) have been linked to the aetiology of schizophrenia through their direct regulation of neurotransmission and immunological pathways that are known to be altered in the disorder (Zhang et al., 2023; Thomas & Zakharenko, 2021). Moreover, dysregulation of some miRNAs has been found not only in the hippocampus, but also in circulating blood vesicles of affected individuals in association with treatment-resistance and the severity of cognitive symptoms (Barnett et al., 2023). However, most of these studies are largely descriptive and none of them has been able to directly assess the potential contribution of sRNAs to the onset and/or progression of the cognitive deficits associated with schizophrenia. Also, genome-wide association studies (GWAS) have found different gene variants associated to cognitive impairment in schizophrenia (Zhao et al., 2022). Some of these risk gene polymorphisms have been located at the forkhead-box P2 (FOXP2) gene, which encodes for a transcription factor involved in regulating synaptic plasticity, neurotransmission and the development of neurons related to language and memory function (Vernes et al., 2007; Lang et al., 2019). FOXP2 has been linked to schizophrenia vulnerability, auditory hallucinations and cognitive deficits in chronic patients with the disorder (Sanjuán et al., 2006; Lang et al., 2019; Sanjuán et al., 2021), although the association of some specific polymorphisms to the disease are still controversial. Furthermore, increased FOXP2 levels have been found in some experimental models of schizophrenia. Nevertheless, it remains to be confirmed whether FOXP2 could mediate the development of the different symptoms associated with schizophrenia and its underlying molecular mechanisms. In this thesis, we hypothesise that sRNAs dysregulated in the hippocampus of patients with schizophrenia are important contributors to its associated cognitive deficits. Furthermore, we aim to decipher whether altered levels of FOXP2 in the brain of these patients could play a role in the onset and progression of some of those clinical features of the disorder. Characterization of hippocampal sRNA profiles from patients with schizophrenia revealed novel microRNA (miRNA) species dysregulated in the disorder. To define the contribution of sRNAs to the cognitive-like symptoms of schizophrenia, we developed a novel translational model based on the injection of those sRNAs isolated from the hippocampus of schizophrenia patients or non-affected individuals into the brain of wild-type mice. Animals receiving sRNAs from diseased patients exhibited an impairment in hippocampal-dependent spatial short-term memory in the T-maze test, in comparison with mice receiving sRNAs from healthy controls or vehicle. However, no deficits were found in recognition memory when performing the novel object recognition (NOR) test, along with no changes in anxiety-like levels nor locomotor activity in the open field test. Golgi staining revealed that schizophrenia sRNAs induced a decrease in the spine density of the pyramidal neurons from the cornus ammonis 1 (CA1) hippocampal region. Injected mice showed higher levels of synaptotagmin 2, a presynaptic marker for inhibitory interneurons. This increase mirrors observations made in patients. Furthermore, we also detected subtle morphological changes in mouse hippocampal microglia in response to sRNAs from affected individuals. We found increased FOXP2 protein levels in the hippocampus and putamen of patients with schizophrenia. These aberrant levels of FOXP2 were recapitulated in the hippocampus, but not in the striatum, of a chronic ketamine mouse model of experimental psychosis. Overexpression of Foxp2 in the hippocampus of wild-type mice using adeno-associated viral (AAV) vectors induced some positive-like symptoms of the disease, such as hyperlocomotion and alterations in navigation in the open field test. No effects in the negative-like symptoms were found in the three-chamber sociability test. Interestingly, Foxp2 overexpression induced cognitive deficits related to an impairment in recognition memory in the NOR test, while we did not find any differences in the spatial short-term memory when performing the T-maze test. Proteomic analysis of the hippocampus of mice overexpressing Foxp2 revealed a reduction in relevant proteins involved in glutamatergic and GABAergic neurotransmission. Positive correlation between FOXP2 and SYT2 protein levels in the hippocampus of affected individuals suggests these changes could be concomitant processes in the disorder. In summary, our results suggest that dysregulated hippocampal sRNAs in schizophrenia are important contributors to its associated cognitive symptoms, while aberrantly increased levels of FOXP2 in the hippocampus of these patients participate in the pathophysiology of the disorder.
Illuminating cAMP signalling in neurodegenerative diseases: from synaptic plasticity mechanisms to therapeutic opportunities
(Universitat de Barcelona, 2025-07-08) Sitjà Roqueta, Laia; Masana Nadal, Mercè; Universitat de Barcelona. Departament de Biomedicina
[eng] With the ageing of the population, neurodegenerative diseases represent a growing public health problem, which could lead to an unsustainable social and economic burden if current trends persist. Surprisingly, most of these diseases have no cure and not even an effective treatment that improves the quality of life of those affected. A distinctive feature of neurodegenerative disorders is synaptic dysfunction, which appears despite the absence of neuronal loss, and is caused by the accumulation of misfolded proteins, which leads to molecular, circuit, and functional alterations (Palop 2006). However, the exact mechanisms that cause these specific alterations are not yet fully understood. This highlights the need to deepen the understanding of these diseases and, even more importantly, the urgent need to develop new therapeutic strategies, potentially based on the modulation of brain plasticity. In this thesis, we have focused on two neurodegenerative diseases: Huntington's disease (HD) and Alzheimer's disease (AD). HD is an autosomal dominant genetic neurodegenerative disorder, characterized by progressive degeneration of the striatal and cortical regions of the brain (Huntington, 2003; Walker, 2007). The disease manifests itself with motor deficits such as chorea, dystonia and lack of coordination, as well as cognitive impairment and psychiatric disorders. HD is caused by a mutation in the huntingtin gene (HTT), with an expanded repetition of the CAG triplet resulting in a mutated form of the huntingtin protein (mHTT), which leads to neurodegeneration (The Huntington's Disease Collaborative Research Group, 1993a). HD is characterized by selective neuronal loss and impaired synaptic plasticity, especially in the cerebral cortex and striatum, where the middle spinous neurons are especially vulnerable (Vonsattel & DiFiglia, 1998). Disruption of the cortex-striatum pathway, which plays a fundamental role in motor and cognitive functions, contributes to the symptoms of the disease (Cepeda et al., 2007). AD, on the other hand, is the main cause of dementia and is characterized by progressive memory impairment, as well as alterations in language, executive function, and visuospatial skills (Scheltens et al., 2021; Stelzmann et al., 1995). AD is classified as familial, caused by mutations in genes such as APP, PSEN1 and PSEN2, with an early onset that accounts for less than 0.5% of cases, and sporadic AD, which accounts for 99.5% of cases and is influenced by genetic, environmental, and lifestyle factors (Bateman et al., 2011; Bertram et al., 2010). AD is characterized by the presence of amyloid plaques, neurofibrillary buds, and neuroinflammation. Synaptic dysfunction, especially in the hippocampus, is directly correlated with the cognitive impairment observed in AD (Querfurth & LaFerla, 2010). Therefore, synaptic plasticity constitutes an early alteration in both diseases, which highlights the need to develop therapeutic strategies that specifically focus on these initial changes. Synaptic plasticity is the mechanism by which synaptic connections in the brain are strengthened or weakened in response to various stimuli. Alterations in this process constitute one of the main characteristics of HD and AD, contributing significantly to the functional deficits observed in these pathologies (J. Y. Li et al., 2003; Selkoe, 2002). In long-term synaptic plasticity, the activation of receptors by neurotransmitters triggers signaling pathways that favor synaptic strengthening, often associated with calcium input. A key component of this strengthening is the insertion of new receptors into the postsynaptic membrane, a process that requires the activity of protein kinases and local protein synthesis. Beyond calcium, another signaling molecule that is fundamental for maintaining plasticity is cyclic adenosine monophosphate (cAMP). cAMP is modulated by the activity of metabotropic receptors and, among multiple targets, activates protein kinase A (PKA), which phosphorylates transcription factors such as CREB, thus initiating the gene expression necessary for long-term synaptic modifications (Benito & Barco, 2010). In addition, the cAMP-PKA signaling pathway is essential for establishing structural changes at synapses, and its activation has been associated with enhanced synaptic plasticity in several brain regions (C. C. Huang & Hsu, 2006; Nguyen & Kandel, 1997). At the same time, astrocytes also contribute to synaptic plasticity. However, the mechanisms by which cAMP participates in synaptic plasticity have not yet been fully understood, nor their specific role in neurons or astrocytes. In fact, alterations in cAMP signaling are increasingly related to aging and neurodegenerative diseases such as HD and HD (Kelly, 2018). Both the cortex-striatum pathway in HD and the hippocampus in HD show disruptions in the cAMP-PKA signaling pathway. In the case of HD, a decrease in cAMP signaling in the cortex and striatum, and an increase in the hippocampus, have been described, although these results remain controversial. On the other hand, in HD it is better established that cAMP signaling decreases in the hippocampus, contributing in a key way to the development of the pathology associated with dementia. In view of the controversies about the role of cAMP in HD, and considering the great importance of the cortex in this disease, our first objective is to characterize the alterations in cAMP signaling and the behavior associated with the cortex of the R6/1 mouse model for the disease. Therefore, strategies aimed at enhancing synaptic plasticity by modulating cAMP signaling could have great potential to mitigate or delay neural network dysfunction associated with neurodegenerative disorders such as HD and AD. In this sense, optogenetic tools allow precise control of biological mechanisms using light-sensitive proteins. In particular, photoactivated adenylate cyclades (PACs) are enzymes that increase cAMP levels in response to light, through an adenylate cyclase domain coupled to a photoreceptor module (Iseki & Park, 2021). Among these, DdPAC is a newly optimized CAP that regulates cAMP levels in response to red light (Stüven et al., 2018). Initially developed in bacteria, DdPAC has demonstrated a more potent light response compared to other red light-sensitive PACs. However, its application in brain cells and in vivo has not yet been explored. Considering the ability of red light to penetrate tissues with minimal dispersion, DdPAC represents a promising tool for non-invasive applications. Therefore, our second objective is to establish the use of DdPAC as an optogenetic tool to modulate synaptic plasticity in vivo, in a non-invasive way. On the other hand, given the regional vulnerability and pathophysiological differences observed between HD and AD, which can affect cAMP modulation and synaptic plasticity, our third objective is to restore physiological function through light stimulation of DdPAC in mouse models of both diseases. In this case, attention will be focused on the regions most affected by each pathology: the striatum and cortex in the case of HD, and the hippocampus in AD. Taking this information into account, the main objective of this thesis is to restore physiological function in neurodegenerative diseases by modulating brain plasticity through light activation of cAMP signaling, mediated by DdPAC, in specific brain circuits To achieve our first objective and characterize alterations in cAMP signaling and cortex-related behavior in the R6/1 mouse model of Huntington's disease, we first evaluated cAMP alterations in the cortex during behavioral tasks related to the cortex-striatum pathway in R6/1 mice. To investigate the dynamics of cAMP, we performed fibre photometry recordings using the GFlamp-1 sensor, a novel cAMP sensor, on neurons in the M2 cortex of 14- and 20-week-old WT and female WT and R6/1 mice, during beetle-mania (BMT) and rotarod accelerator (ARR) tasks. We first evaluated the dynamics of cAMP during BMT, observing an increase in cAMP levels in both WT and R6/1 mice after beetle introduction. Although the R6/1 mice already showed altered behaviour during the test, no differences in cAMP levels were detected between the genotypes. These data reveal the involvement of neuronal cAMP signaling during BMT, with minimal alterations in R6/1 mice. Subsequently, to better understand the contribution of cAMP in tasks related to the M2 cortex, we explored the dynamics of cAMP during ARR. Our results were in line with those observed during BMT, as neuronal cAMP levels in both WT and R6/1 mice increased with the onset of the task. Unlike the results obtained with BMT, in this case we observed an aberrant over-activation of the M2 cortex in R6/1 mice. Finally, to find out if alterations in cAMP activity during M2-related tasks are more evident in more advanced stages of the disease, we repeated BMT in the same cohort of mice at 20 weeks, when the animals are fully symptomatic. At this age, we could still observe an increase in neuronal cAMP in WT and R6/1 after the introduction of the beetle. However, this increase was significantly smaller in R6/1 mice. Overall, these results highlight the involvement of neuronal cAMP in tasks related to the M2 cortex and show alterations in the context of HD. Given the critical importance of the M2 cortex in the pathophysiology of HD, we also wanted to determine whether additional symptoms associated with cortex-striatum dysfunction arise in the early stages of the disease in the R6/1 model. To do this, we selected two behavioral tests, the adhesive removal test and the marble-burying test, both related to the M2 cortex and the cortex-striated pathway, and carried them out longitudinally from 4 to 16 weeks of age. In the adhesive removal test, related to the M2–somatosensory cortex–striatum pathway, we observed motor deficits from 8 weeks, while somatosensory deficits appeared at 16 weeks. In the marble-burying test, related to the M2–orbitofrontal–striated cortex pathway, we observed a similar behaviour to anhedonia from 8 weeks onwards. These data indicate that the dysfunction of the cortex-striatum pathway arises in very early stages, highlighting the potential of early therapeutic interventions. Overall, the results of this first objective indicate that crust-striatum pathway dysfunction related to the M2 cortex emerges in the early stages of the disease, highlighting the potential of therapeutic interventions in early stages. Since neuronal cAMP signaling remains functional in mice with altered behavior, it is unlikely to be directly responsible for M2-related behavioral deficits, suggesting that other mechanisms could be involved. To achieve the second objective and implement DdPAC as a new optogenetic tool to modulate synaptic plasticity in a non-invasive way through cAMP signaling, we first wanted to establish a minimally invasive method for its administration to the brain through AAV vectors. For this reason, we designed viral constructs with GFP under three different promoters (CAG, CamKIIa and FLEXon), and administered them using two AAV serotypes (AAV9 and PHP.eB), in two different mouse strains (C57BL/6J and B6CBA), and through three routes of administration, from more to less invasive (intra-cranial, retro-orbital and intra-nasal). In summary, GFP expression was detected in several brain regions and in specific cell types after retro-orbital injection of the PHP.eB and AAV9 vectors, showing the PHP.eB serotype a wider infection. In addition, we managed to express the viral constructs of PHP.eB in two murine strains and specifically in our R6/1 model. In addition, retro-orbital injection into A2a-Cre mice resulted in specific regional transduction, demonstrating its potential to direct specific circuits. However, no GFP fluorescence was observed after intra-nasal administration in any of the cases. These results highlight retro-orbital injection as a minimally invasive pathway to reach brain regions efficiently and in different cell types and mouse strains, offering an alternative to stereotactic surgery. However, further research is needed on viral capsid modification to facilitate neural cell infection via intranasal administration. To continue with this objective, we characterized the effects of DdPAC activation on specific cell types in the brain. First, we investigated whether cAMP modulation by DdPAC was able to promote synaptic plasticity. Therefore, we injected DdPAC under the CamKIIa or GFAP promoters into the cortex to selectively express it in neurons and astrocytes, respectively, and subsequently made recordings with multiple electrode arrays (MEAs). Red light illumination succeeded in enhancing neuronal activity in both neuronal and astroglial activation of DdPAC, although the effect was more pronounced when it was activated in astrocytes. For this reason, subsequent experiments with this objective focused on investigating the activation of DdPAC in cortical astrocytes. Thus, we characterized the underlying mechanism of this potentiation, demonstrating that the DdPAC-induced cAMP increase is PKA and NMDAR dependent, but calcium-independent, requires synaptic activity, and induces glutamate gliotransmission. To delve into the in vivo effects of astroglial activation of DdPAC, we performed phosphoproteomics and proteomics analyses. The omics data validated the involvement of the cAMP-PKA pathway in the astroglial effects of DdPAC, supporting its central role in synaptic plasticity, while revealing a broad brain effect derived from astrocyte activation. Overall, the data from the second objective position DdPAC as a powerful tool to modulate synaptic plasticity in the brain through targeted manipulation of the cAMP-PKA pathway in astrocytes, while establishing a robust and minimally invasive method for its application in vivo. Finally, we addressed our third objective: to restore physiological function through light stimulation of DdPAC in mouse models of HD and AD. First, we investigated the functional effects of astrocytic activation of DdPAC in two of the most affected regions in HD: the cerebral cortex and striatum. To do this, we injected DdPAC into cortical or striatal astrocytes of the R6/1 mouse model to evaluate their ability to modulate brain function. In cortical stimulation experiments, hemodynamic changes were analyzed using an imaging technique based on light scattering and, subsequently, motor behavior was evaluated. Hemodynamic analysis revealed an over-activation of the cortex after acute activation of DdPAC in astrocytes in the cortex of R6/1 mice, a response that was not observed in control mice. In addition, repeated stimulation of DdPAC in astrocytes in the cortex impaired coordination in R6/1 mice, as evidenced in the vertical pole test, while motor learning improved in WT mice, assessed by the accelerated rotarrod test. In addition, post-mortem analysis revealed an increase in GFAP expression only in WT mice after repeated stimulation and behavioral testing. Next, we examined motor behavior after stimulation of DdPAC in striatal astrocytes. In this case, the modulation of cAMP by DdPAC in astrocytes impaired coordination in both WT and R6/1 mice, while motor learning remained preserved. In addition, GFAP expression increased in both groups. Taken together, these results suggest that astrocytic modulation of DdPAC produces differentiated effects according to brain region and molecular context, with diverse results in the cortex and striatum in WT and R6/1 mice. In addition, our data suggest that increased cAMP levels in astrocytes may be detrimental in the context of HD, so strategies aimed at specifically reducing astrocytic cAMP could have more therapeutic potential. In parallel, we investigated the effects of cAMP signaling by DdPAC on neurons and astrocytes of the hippocampus, the most affected region in AD. To do this, we injected DdPAC into neurons and astrocytes in the hippocampus of WT and 5xFAD mice, followed by histological and proteomic analyses. Notably, histological analyses revealed a reduction in GFAP expression and amyloid-β deposits in the hippocampus where DdPAC had been activated in astrocytes, but not in regions with neuronal activation. Proteomic analysis of DdPAC-activated astrocytes revealed a response mainly associated with glial activation and immune response processes, as well as synaptic plasticity, while neuronal activation mostly influenced synaptic plasticity. In both cases, cytoskeletal regulation emerged as a key function, although the proteins involved differed between neurons and astrocytes. In addition, the effects of DdPAC activation, both in neurons and astrocytes, varied between WT and 5xFAD mice, indicating a differential effect depending on the molecular context. These results reinforce the idea that the effects of modulation by DdPAC depend on the brain region, molecular context and cell specificity. In summary, this thesis provides new knowledge about the cAMP pathway in synaptic plasticity and neurodegenerative diseases. First, our data demonstrate the participation of neuronal cAMP in behaviors related to the M2 cortex and reveal alterations in the dynamics of neuronal cAMP in mice with HD. In addition, we identify early motor and psychiatric deficits associated with the M2 cortex, highlighting the potential for therapeutic interventions in the early stages. To facilitate the application of therapies in the brain, we established a minimally invasive method for the administration of AAV that allows effective expression of the transgene in specific cell types and murine strains. Notably, we have demonstrated the ability of cAMP modulation by DdPAC to enhance synaptic plasticity in the cortex, especially when activated in astrocytes. In the context of neurodegeneration, the activation of DdPAC in HD and AD models produces differential effects: in HD, the activation of cortical astrocytes improves motor learning in WT mice but impairs coordination in mice with HD, while the activation of striatal astrocytes negatively affects coordination in both. In AD, increased cAMP in hippocampal astrocytes reduces astrogliosis and amyloid-β deposits, while neuronal activation reduces microglial reactivity. Proteomic analysis of hippocampal samples reveals differential changes induced by DdPAC in neurons and astrocytes, as well as between WT and AD mice, linking the activation of the cAMP-PKA pathway to synaptic plasticity and immune responses. In conclusion, this thesis reveals new functions of cAMP signaling and proposes it as a promising therapeutic target in neurodegenerative diseases. Our results demonstrate that the modulation of cAMP by DdPAC in neurons and astrocytes has a profound impact on neuronal plasticity, with the modulation of cAMP-PKA in astrocytes producing the broadest effects. In addition, we highlight the importance of the brain region and the molecular context in the results of cAMP modulation, since the activation of DdPAC produces differentiated effects in the cortex, striatum and hippocampus, and according to the pathological state. Ultimately, we provide strong evidence that positions DdPAC as a versatile tool to modulate cAMP-PKA signaling, with potential applications in both neuroscience research and therapeutic development.
Development of an in vitro LTP-model of food anaphylaxis and study of mechanisms driving exacerbated responses
(Universitat de Barcelona, 2025-06-19) Ollé Boix, Laia; Martín Andorrà, Margarita; Muñoz-Cano, Rosa Maria; Universitat de Barcelona. Departament de Biomedicina
[eng] Mast cells (MCs), key immune system cells, can be activated via IgE or the MRGPRX2 receptor, releasing proinflammatory mediators that may trigger severe allergic reactions such as anaphylaxis. The study aims to better understand the cellular and molecular mechanisms governing these responses and to identify potential therapeutic targets. This study develops an in vitro model of food allergy mediated by lipid transfer protein (LTP), focusing on the analysis of mast cell (MC) responses in patients with varying degrees of severity. Patients sensitized to Lipid transfer protein (LTP) were classified as anaphylaxis or sensitized depending on the symptoms elicited by LTP-containing food. CD34+-derived MCs from patients and controls were obtained, sensitized with pooled sera, and challenged with Pru p 3 (peach LTP). MCs from anaphylactic patients exhibited increased degranulation and elevated secretion of PGD2, IL-8, and GM-CSF upon stimulation, which could be related to higher-affinity IgE, produced with the help of TFH13 cells, which were more abundant in those patients. In contrast, sensitized MCs showed a protective profile, with higher CCL2 and TGF-β production. At the molecular level, MCs from anaphylactic patients expressed higher levels of genes associated with cell activation, inflammation, and mitochondrial function, including the Microphthalmia-associated transcription factor (MITF). Further dissection shows that inhibition of MITF significantly reduced degranulation, calcium influx, mediator secretion, and mitochondrial activity. In conclusion, allergic response severity is influenced by both humoral and cellular components. Beyond antibody-driven mechanisms, cellular factors such as MITF expression may amplify mast cell activation and exacerbate allergic reactions. Targeting MITF could represent a therapeutic strategy to reduce mast cell hyperactivation and mitigate severe allergic responses.
Unraveling the neuro-immunological implications of the Ikaros family in schizophrenia and neuroinflammation
(Universitat de Barcelona, 2025-05-06) Ballasch, Iván; Giralt Torroella, Albert; Alberch i Vié, Jordi, 1959-; Universitat de Barcelona. Departament de Biomedicina
[eng] The Ikaros family of transcription factors has been largely described in the context of immune system function and immune system cells development. In the last years, some of its members were found in neural cells, particularly during perinatal life, associated with CNS development and proper function of some brain regions, such as the hippocampus. Dysregulations of the Ikaros family members have been linked to a wide range of immune-related disorders. However, thei mplication of the Ikaros family in brain-related disorders remains a widely underexplored area. Schizophrenia is a psychiatric condition affecting around 21 million people worldwide with an annual associated cost of around 16.500€ per patient in Europe. Schizophrenia symptoms are classified into three broad categories: Positive (e.g. hallucinations, delusions and thought disorder), Negative (e.g. withdrawal and lack of motivation) and Cognitive (e.g. deficiencies in executive functions and working memory). Some of the key-affected brain regions in schizophrenia are the pre-frontal cortex and the hippocampus. Genetic and environmental factors play a role in the risk of developing this condition and, in many cases, these factors act through the immune system. Indeed, one of the most prominent discoveries in schizophrenia research in the last years has been the association of immune dysfunction with its pathogenesis, proposing an aberrant neuro-immune crosstalk in this condition. In that context, two major ways the immune system is proposed to contribute to the appearance of schizophrenia are: neuroinflammation and T cells dysfunction. Microglia cells are one of the main populations orchestrating neuroinflammation processes within the brain. In schizophrenia, microglia density and cell number have been shown to be generally increased while alterations in density and number of other glia cells seem to be minor. Besides, many pro-inflammatory cytokines, such as IL-6 or TNF-α, have been found to be increased in schizophrenia patients’ post-mortem brains. On the other side, T cell mediated immunity dysregulations have also been proposed in schizophrenia, with abnormal invasion of T cells within patients’ brains and possibly alterations of dopamine-T cells crosstalk. Therefore, T cells population could be considered as a candidate mediating the aberrant neuro-immune crosstalk in schizophrenia possibly involving neurotoxic effects and contributing to neuroinflammation. Previous studies have shown that Ikaros and Helios are essential for the proper function of some immune cell types. On the other hand, SPNs of IKAROS gene have been found to be related to the age of onset of schizophrenia and Helios protein deficiency have been shown to alter molecular pathways which are also altered in schizophrenia. Furthermore, preliminary data from our lab indicated that Ikaros and Helios are dysregulated in immune cells of schizophrenia patients. Consequently, we aimed to evaluate the potential involvement of Ikaros and Helios in the pathophysiology of schizophrenia. In the first article, we have found downregulated levels of Ikaros and Helios in PBMCs of schizophrenia patients while their levels were not different from controls in post-mortem brain samples. A doble mutant animal model mimicking the double downregulation of Ikaros and Helios also showed schizophrenia-like behavior in the three categories of symptoms. Using the secretome of the PBMCs from schizophrenia patients, we induced several schizophrenia-like phenotypes in in-vitro and in-vivo translational models. We have characterized the molecular profile of the PBMCs secretome and we identified IL-4 and CXCL10 as possible candidates mediating its effects. In the second article, we have identified Ikaros as a factor involved in microglia homeostasis, particularly in inflammation-associated conditions. Using an Ikaros total Knockout model we identified deficiencies linked to a hippocampal-dependent task. Thereafter, we focused on the hippocampus. The Ikaros KO model presented microglial morphological changes associated to inflammatory conditions such as elevated expression of TNFα and NF-kβ and long-term potentiation defects. Besides, we tested the level of Ikaros in other conditions of neuroinflammation and we found augmented levels of Ikaros in all of them. Correspondingly, microglia phagocytic activity was also disrupted under the modulation of Ikaros in vitro. The elevated levels of some inflammasome members and alterations in the acetylation-methylation ratios found in the KO model give some ideas about the mechanisms by which Ikaros could modulate microglia function. The final model about how the downregulation of Ikaros and Helios in PBMCs of schizophrenia patients could be contributing to the pathogenesis of the condition could be summarized as follows: The combination of the downregulations of Ikaros and Helios in different PBMCs subsets possibly generates elevated levels of IFN-γ and reduced immune suppression capacity of Treg cells. IFN-γ is priming microglia cells that upon second immune stimuli generate oversimplified and neurotoxic inflammatory responses over years deteriorating the nervous tissue until the manifestation of the first psychotic episode. A possibility is that Ikaros dysregulation within microglia is exacerbating this circuit.
Characterization of striatal development in Huntington’s Disease and its implication to the adult phenotype
(Universitat de Barcelona, 2023-09-15) Vila Torondel, Cristina; Canals i Coll, Josep M.; Universitat de Barcelona. Departament de Biomedicina
[eng] Huntington's disease (HD) is a devastating neurodegenerative disorder that manifest itself through motor, cognitive and behavioral symptoms. HD is a monogenic disease caused by CAG expansion in the huntingtin gene, which inversely correlates with the age of onset. Despite the ubiquitous expression of mutant huntingtin (mHtt), striatal-projection neurons, known as medium spiny neurons (MSN), are the most vulnerable cell type. Although the onset of the disease has been classically defined by the appearance of motor symptoms, which occur around the age of 40-50, recent studies have shown significant cognitive and mental dysfunction many years before motor onset. A key role of huntingtin in brain development has been described. For this reason and given that the mutation exists from the moment of conception, hypothesis about a possible developmental effect on HD started to emerge. Subtle effects during development can lead to deficiencies in the cellular homeostasis of evolving regional neuronal subpopulations, which in turn can result in adult- onset cell death by normally non-lethal environmental stressors. Neurodegenerative diseases can therefore represent an emerging class of developmental disorders characterized by little developmental abnormalities that cause no obvious deficits. In this thesis we have studied and characterized these developmental alterations in a model of the disease, the zQ175 mouse. Our results show that mouse embryos that have inherited the mutation present a gene expression profile related to the pathophysiology described in adult patients with the disease, from early stages of development. In addition, we have detected that HD developmental alterations are specific to neuronal subpopulation. During the development of the mouse striatum, indirect pathway MSNs (iMSNs) are generated prematurely and excessively during the period of neurogenesis. However, these neurons exacerbately die during the natural period of programmed cell death, which occurs in postnatal stages. Thus, the striatal development of HD mice ends up with a striatum with a lower proportion iMSNs, generating an imbalance between their main neuronal populations. In addition, single-cell analysis allowed us to identify a specific neural progenitor cell (NPC) population differentially affected during HD development. The identification of subtle changes in specific populations of NPCs, and their subsequent follow-up to adults, may clarify neurodevelopmental's contribution to HD. These results suggest that the subtle effects of the mutant huntingtin during development may cause significant alterations that spread to adults and contribute to the most common HD phenotypes.
Role of VPS13A in corticostriatal synaptic transmission in a model of Chorea-Acanthocytosis
(Universitat de Barcelona, 2023-07-25) García García, Esther; Rodríguez Allué, Manuel José; Masana Nadal, Mercè; Universitat de Barcelona. Departament de Biomedicina
[spa] El objetivo principal de esta tesis ha consistido en analizar el papel de VPS13A en la plasticidad sináptica y el comportamineto corticostriatal en un modelo murino de corea-acantocitosis. En primer lugar hemos demostrado que VPS13A se expresa en el cerebro de ratón desde las etapas embrionarias y que es estable a lo largo del tiempo. Además VPS13A se distribuye ampliamente a lo largo del cerebro de ratón, con una expresión prominente en la corteza motora, el hipocampo, el puente troncoencefálico y el cerebelo. Por último, hemos demostrado que VPS13A es expresada principalmente por neuronas, localizada en la región perinuclear, y presente pero no enriquecida en los sinaptosomas. En segundo lugar hemos gerenado un modelo murino mediante la reducción de la expresión de VPS13A neuronal (VPS13A-KD) tanto in vitro como in vivo. Hemos demostrado que la reducción de VPS13A en la vía corticostriatal modifica la locomoción y el comportamiento exploratorio in vivo y altera la depresión corticostriatal a largo plazo ex vivo. Además, la reducción de VPS13A en la vía corticostriatal no induce pérdida neuronal pero sí que desencadena una reacción microglial en el cuerpo estriado. Por último, nuetros resultados muestran que la reducción de VPS13A en la vía corticostriatal altera la expresión únicamente de marcadores pre-synapticos y modifica la señalización tanto del factor neurotrófico derivado del cerebro (BDNF) como de la fractalkina (CX3CL1). Finalmente, hemos demostrado que VPS13A se encuentra en los sitios de contacto entre retículo endoplasmático y mitocondrias y que la reducción de VPS13A in vitro, induce estrés mitocondrial. En resumen, en esta tesis demostramos que VPS13A tiene un papel relevante en la plasticidad y conectividad sináptica corticostriatal mediante su participación en la señalización de BDNF, en el control neuronal de la poda y plasticidad sináptica mediada por microglia y en el mantenimiento de la homeostasis mitocondrial.
Unravelling the contribution of small non-coding RNAs to Huntington’s disease pathogenesis
(Universitat de Barcelona, 2023-07-21) Guisado Corcoll, Anna; Pérez Navarro, Esther; Martí Puig, Eulàlia; Universitat de Barcelona. Departament de Biomedicina
[eng] BACKGROUND AND OBJECTIVES: Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an abnormal expansion in the number of CAG triplets located at exon 1 of the huntingtin gene (HTT), resulting in an expanded portion of glutamines (polyQ) in a region near the amino terminal end of the HTT protein (The Huntington's Disease Collaborative Research Group, 1993). This expansion contributes to the formation of an aberrant and misfolded protein that has been associated with aggregation and toxicity (Labbadia & Morimoto, 2013), both of which are mechanisms that have been implicated in multiple neurological disorders. In addition, although this mutation is ubiquitous in all cells of the body, selective vulnerability has been described on the part of medium-sized spinous neurons (MSNs) located in the striatum nucleus, formed by the caudate and putamen in humans (Vonsattel et al., 1985). These striatal MSNs can be classified into two types depending on the receptors they express and the brain areas they project to (Gerfen et al., 1990), forming the direct and indirect pathways of the basal ganglia. The correct balance of signaling between these pathways is what allows the voluntary control of movements, so that striatal atrophy, preferably of the indirect pathway at the beginning, during the progression of the disease leads to an alteration in the circuits giving rise to the motor symptoms characteristic of HD (Albin et al., 1989). To date, most of the research on the molecular mechanisms involved in the pathophysiology of HD has focused on the alterations caused by the mutated protein (Zuccato et al., 2010). However, in recent years, it has been observed that the toxicity generated by the mutated protein coexists with RNA-mediated pathogenic mechanisms, which can be subdivided into those associated with expanded RNA with CAG repeats and those related to other non-coding RNAs. On the one hand, it has been observed that RNA with CAG triplet expansion can exert its toxicity through multiple mechanisms (Heinz et al., 2021; Malik et al., 2021; Martí, 2016). Among them, we find the formation of short-sized RNAs (21 nt) with CAG repeats (sCAG) through Dicer, whose levels are dependent on the length of triplet expansion (Bañez-Coronel et al., 2012). These species have gene silencing capacity in such a way that they have been associated with transcriptomic alterations and can affect neuronal viability per se (Bañez-Coronel et al., 2012). In addition, our group has also described that its effects can be partially reversed with the use of a modified antisense oligonucleotide, targeting CAG repeats (LNA-CTG), producing a recovery of motor function and levels of multiple striatal markers in a murine model of HD (Rué et al., 2016). On the other hand, in recent years, different mechanisms of RNA toxicity involving multiple biotypes of non-coding RNAs have also been described sRNA, <200 nt), such as microRNAs (miRNA), ribosomal RNAs (rRNA) or transfer RNAs (tRNAs), which have also been clearly associated with the regulation of gene expression, among many other functions (Cech & Steitz, 2014). At the same time, it has been observed that in the context of HD there is significant transcriptomic dysregulation in different brain regions (Martí et al., 2010), pointing to a possible role of sRNAs in the pathophysiology of HD. In addition, although the canonical function described by tRNAs is based on allowing the synthesis of proteins in the ribosome by decoding information at the mRNA level, implications of tRNAs in other biological processes such as cell signaling and survival, apoptosis, amino acid metabolism or even in stress response programs have also been described (Raina & Ibba, 2014). Specifically, many of these functions have been attributed to fragments derived from these tRNAs (tsRNAs), some of which are constitutive components of all cells, while others only occur in situations of cellular stress (Anderson & Ivanov, 2014) and have been related to different pathological situations such as cancers, infections and different paradigms of neurodegeneration (R. Magee & Rigoutsos, 2020). Based on the bibliographic information presented, our hypothesis is that the pathogenic mechanisms caused by RNA, and specifically sRNA, are much more involved than previously thought in HD and that their study becomes of high importance both to understand the pathophysiological bases of HD and to develop new therapies. Therefore, the main objective of this project was to identify new species of sRNA involved in the pathogenic mechanisms of HD and determine their contribution to the alterations characteristic of this pathology.
Role of MITF in IgE and MRGPRX2-dependent mast cell activation
(Universitat de Barcelona, 2023-07-20) Guo, Yanru; Martín Andorrà, Margarita; Universitat de Barcelona. Departament de Biomedicina
[eng] Anaphylaxis is a severe allergic reaction triggered by allergens to certain foods, medicines, insect venom, and other substances. Anaphylactic shock can be fatal if not treated. Mast cells are the effector cells in anaphylaxis, and mast cell transcriptional factors are crucial in controlling the production of mediators. In summary, this thesis shows the relevance of microphthalmia associatedtranscription factor (MITF) in IgE and MRGPRX2-dependent mast cell activation. Thus, MITF and MITF-dependent genes may become targets to treat IgE or MRGPRX2-dependent pathologies.
Inmunoterapia del cáncer colorrectal mediante la combinación de fármacos bloqueantes de la vía PD-1/PD-L1 y vacunas de células dendríticas autólogas
(Universitat de Barcelona, 2023-07-20) Español Rego, Marta; Benítez-Ribas, Daniel; Universitat de Barcelona. Departament de Biomedicina
[spa] En esta tesis se estudia el cáncer colorrectal metastásico y posibles alternativas de tratamiento. Se plantea una terapia combinada con células dendríticas autólogas e inhibidores de la vía PD-1/PD-L1 y se profundiza en el microambiente de este tipo de tumores. Se describe un estudio clínico en el cual se tratan por primera vez pacientes con cáncer colorrectal metastático de tipo MSS con una combinación de células dendríticas y avelumab, un inhibidor de PD-L1. Este grupo de pacientes responden muy mal a la terapia con checkpoint inhibitors (y a terapia convencional), con lo que es necesario explorar otras vías de tratamiento más eficaces. Las células dendríticas han mostrado resultados clínicos limitados, aunque han demostrado capacidad para estimular a los linfocitos T antitumorales, con lo que esta combinación se plantea como una estrategia con capacidad para vencer esta resistencia tumoral, combinando la capacidad de las células dendríticas de estimular a los linfocitos T junto con al capacidad del avelumab para frenar la inhibición de estos mismos linfocitos. En este estudio también se plantea una monitorización exhaustiva de la respuesta inmune de los pacientes con el objetivo de encontrar biomarcadores de respuesta al tratamiento. A pesar de que los resultados clínicos del estudio han sido más modestos de los esperados, la terapia ha mostrado ser segura y factible, y gracias a las monitorización de los pacientes pre y post tratamiento se ha podido describir por primera vez una remodelación metabólica post tratamiento, lo cual plantea nuevas posibilidades para diseñar terapias dirigidas en este grupo de pacientes, y se han encontrado posibles biomarcadores de respuesta al tratamiento para poder seleccionar a pacientes candidatos a este tipo de terapia.
Insights in the pathogenesis of the aortic aneurysm in Marfan syndrome and new therapeutic approaches
(Universitat de Barcelona, 2023-07-20) Rodriguez Rovira, Isaac; Egea Guri, Gustavo; Universitat de Barcelona. Departament de Biomedicina
[eng] The objective of this thesis was to investigate the effect of a potent inhibitor peptide (P144) targeting transforming growth factor β (TGFβ) on the development of aortic aneurysms in a mouse model of Marfan syndrome. We also aimed to assess the role of oxidative stress and hyperuricemia in the progression of aortic aneurysms in Marfan syndrome. To investigate the effect of P144, a chimeric gene encoding the P144 peptide linked to apolipoprotein A-I was delivered using a hepatotropic adeno-associated vector. Two experimental approaches were employed: a preventive treatment administered before the onset of aortic aneurysms and a palliative treatment administered once the aneurysm was already formed. The preventive treatment with P144 successfully prevented the onset of aortic dilation and improved the morphology of elastic fibers and normalized TGFβ signaling. However, the palliative treatment did not halt the progression of the aneurysm. Furthermore, we examined the role of oxidative stress and hyperuricemia in aortic aneurysm development. We found increased production of reactive oxygen species (ROS) and upregulation of XOR, an enzyme involved in ROS production, in both human Marfan syndrome patients and mouse models. Treatment with the XOR inhibitor allopurinol effectively halted the progression of aortic aneurysms in Marfan syndrome mice and mitigated associated endothelial dysfunction and collagen remodeling. Additionally, we investigated the association between hyperuricemia and aortopathy in Marfan syndrome mice and found that hyperuricemia did not exacerbate or alleviate aortic aneurysm development. The findings of this study suggest that P144 is effective in preventing the onset of aortic aneurysms by reducing excessive TGFβ signaling. Oxidative stress plays a role in the formation and progression of aortic aneurysms in Marfan syndrome, and treatment with allopurinol as an antioxidant can interfere with the progression of aortic aneurysms. However, hyperuricemia does not interfere with the development of aortic aneurysms or cardiopathy in Marfan syndrome mice. These results highlight the importance of targeting TGFβ signaling and oxidative stress during the early stages of aortic disease progression in individuals with Marfan syndrome.
Progressive hippocampal alterations in stress-induced major depression and novel therapeutic strategies
(Universitat de Barcelona, 2023-06-30) Sancho Balsells, Anna; Giralt Torroella, Albert; Universitat de Barcelona. Departament de Biomedicina
[eng] Major depression disorder (MDD) is a serious, frequent, and recurrent mental illness that affects millions of people worldwide. One of the most important environmental risk factors associated with DM is chronic stress. Although stress and depression affect various brain regions, the hippocampus plays a key role in this pathology. Due to the pathophysiological complexity of DM, current treatments are ineffective in 30% of patients and are often associated with significant side effects. Therefore, the design of more effective therapeutic strategies is necessary. Hence, in this thesis we wanted to elucidate the effects of stress in mice, determine pathological mechanisms in the hippocampus and modulate them using different strategies. Firstly, the results obtained have shown that the effects of stress on behavior is progressive, since prolonged periods of stress are needed to see a robust behavioral effect. We then determined which specific neuronal subtypes play a fundamental role in the stress response. The results obtained have identified that CA1 of the hippocampus is highly sensitive to the duration of stress and that Egr1 in superficial pyramidal neurons plays a crucial role in the sequelae induced by chronic stress. In addition, proteomic studies performed in the hippocampus have highlighted that Sirt1 is a complex and interesting molecule involved in depression caused by chronic stress, the levels of which are increased with stress. In addition, in this thesis we tried to modulate the microbiota-intestine-brain axis through combined photobiomodulation. The results obtained have shown that simultaneous photobiomodulation of the abdomen and head is beneficial in chronically stressed mice as it produces improvements at the behavioral level, in dendritic spines, in microbiota dysbiosis and in Sirt1 levels, among others. Finally, we determined the role of GSK3β in depression caused by chronic stress. The results obtained demonstrate that the inhibition of GSK3β through the administration of meridianins, a marine natural product with bioactive properties, is beneficial as it induces behavioral improvements. In conclusion, the hippocampal pathogenic mechanisms demonstrated in this thesis provide a new understanding of the pathophysiology of stress-induced major depression. In addition, we propose several cutting-edge strategies to try to reverse stress- induced alterations at different levels: behavioral, cellular, and molecular.
The role of 4E-BP1 in Huntington’s disease: modulation of translation initiation as a therapeutic strategy
(Universitat de Barcelona, 2023-06-21) Castany Pladevall, Carla; Pérez Navarro, Esther; Universitat de Barcelona. Departament de Biomedicina
[eng] Precise regulation of translation initiation plays a critical role in learning and memory processes, and defects in this process have been linked to numerous cognitive disorders including neurological disorders such as addiction, fragile X syndrome or autism, as well as various neurodegenerative disorders such as Parkinson's disease. Previous results from our group described the increase in the formation of the eIF4F protein complex and the inactivation of factor 4E-BP1 accompanied by the aberrant increase in the de novo translation rate in the striatal neurons of the R6/1 mouse, a model of Huntington's disease (HD). In addition, we observed that intracerebroventricular injection of the inhibitor of the initiation complex 4EGI-1 is able to normalize protein synthesis and improve motor symptomatology in this HD model. For this reason, in this Thesis, we have continued to explore the contribution of increased cap-dependent translation to HD progression. Specifically, we have evaluated the potential of modulation of 4E-BP1 factor activity, as a key limiting step in the initiation of cap-dependent translation, in the reduction of striatal pathology that occurs in HD. Second, we have established the primary cultures of striatal neurons from the R6/1 mouse as an in vitro model capable of replicating the same translational alterations observed in the striatum and have used it to identify the potential of generic drugs already approved by the FDA and widely distributed (i.e. sertraline and metformin) to normalize the increase in cap-dependent translation in the molecular context of HD. In addition, we have explored the potential of the selected drugs to normalize the increase in cap-dependent translation and to improve the symptoms by reproducing the effect observed with the intracerebroventricular injection of the inhibitor 4EGI-1, which is still an experimental drug, in R6/1 mice. Finally, we have described alterations in translation in the hippocampus of R6/1 mice including an increase in phosphorylation of factor 4E-BP1 and de novo synthesis. Specifically, we have observed that translation dysregulation selectively affects proteins of pathways associated with energy production previously related to the hyperphosphorylation of 4E-BP1. In addition, the normalization of protein synthesis through intracerebroventricular injection of 4EGI-1 has also induced an improvement in cognitive symptomatology associated with the hippocampus, reinforcing the hypothesis of dysregulation of the translation initiation process as a molecular mechanism and potential therapeutic target in HD.
Uncovering new therapeutic strategies for motor and cognitive deficits in Huntington's Disease
(Universitat de Barcelona, 2023-06-23) Espina Cortes, Marc; Ginés Padrós, Silvia; Universitat de Barcelona. Departament de Biomedicina
[eng] The present thesis focuses on studying possible new therapeutic strategies for the treatment of cognitive and motor symptoms associated with Huntington's disease (HD). More specifically, the first work focuses on studying the contribution of the endoplasmic reticulum (ER) stress to the cognitive deficits of the disease, while the second one studies the role of the ARMS protein in the alterations of the astrocytic secretome and its impact on motor symptoms.
Network alterations in Huntington’s disease beyond the classical model of basal ganglia circuitry
(Universitat de Barcelona, 2023-06-09) Conde-Berriozabal, Sara; Masana Nadal, Mercè; Alberch i Vié, Jordi, 1959-; Universitat de Barcelona. Departament de Biomedicina
[eng] The main objective of this doctoral thesis has been to elucidate the alterations in the dynamics of neural networks in Huntington's disease (HD) beyond the basal ganglia circuit in order to define new therapies based on modulation of the activity of specific circuits. First, we have shown that the secondary motor cortex (M2) and the dorsolateral region of the striatum (DLS) are functionally disconnected in the mouse model of HD R6/1. In addition, we have shown that repeated selective stimulation of this pathway (M2-DLS) improves motor symptomatology and stereotypic behavior, as well as restores synaptic plasticity in mice with HD. Secondly, we have confirmed that the projections of the M2 cortex projecting into the superior colliculus (SC) are structurally and functionally altered. These are accompanied by alterations in the behavior of innate response to threatening visual stimuli, associated with the function of the CS, before motor symptoms appear, both in females and males. Our results suggest that the mechanism underlying these non-motor symptoms could be a lack of activation by the M2 cortex in the face of visual stimulus. Finally, we have demonstrated greater deficits in the functional connectivity of DLS with the rest of the brain than of DMS, and that alterations in motor learning in mice with HD are accompanied by an increase in aberrant neuronal activity by the direct pathway from DLS. In addition, our results show that locomotion and motor learning, both altered behaviors in HD, can be modulated in control mice by selective optogenetic activation of direct or indirect route, respectively. In summary, in this thesis we evidence alterations in the dynamics of neural networks beyond the basal ganglia circuit in the pathophysiology of HD and suggest that the M2 cortex has a key involvement in both motor and non-motor symptoms.
Clasificación automática de malaria e inclusiones eritrocitarias anómalas en sangre periférica utilizando modelos de machine learning
(Universitat de Barcelona, 2025-03-03) Molina Borrás, Angel; Merino, Anna (Merino González); Rodellar, José; Universitat de Barcelona. Departament de Biomedicina
[spa] Los especialistas de laboratorio juegan un papel esencial en el diagnóstico del paludismo mediante la identificación de características morfológicas cualitativas de los hematíes en la revisión del frotis de sangre periférica. Este análisis visual conlleva un consumo de tiempo, requiere un personal bien entrenado, y es sensible a la variabilidad entre observadores. . El objetivo general de esta tesis doctoral es el reconocimiento automático de hematíes parasitados con malaria, así como su diferenciación no solo de hematíes normales sino también de hematíes con otros tipos de inclusiones, mediante herramientas de inteligencia artificial. La primera parte de este trabajo comienza con el desarrollo de un algoritmo de segmentación de hematíes a partir de imágenes de sangre periférica obtenidas con microscopio y el desarrollo de un modelo de reconocimiento automático basado en aprendizaje automático (machine learning) a partir de la extracción de descriptores cuantitativos de las imágenes segmentadas. En una segunda parte de esta tesis, el reconocimiento automático de la malaria se aborda a través de modelos basados en redes neuronales convolucionales. En último lugar, se desarrolla un modelo para la identificación de malaria basado en redes neuronales convolucionales, pero en este caso, mediante el uso de imágenes de sangre periférica obtenidas con el analizador CellaVision. En comparación con el actual estado del arte, esta tesis doctoral supone un avance significativo en la implementación de modelos de reconocimiento automático de malaria en un entorno clínico realista. Esto es posible principalmente al haber incluido en los modelos de clasificación otras inclusiones eritrocitarias que tienen gran similitud morfológica con los parásitos de la malaria. Con esta estrategia ha podido evitarse el alto número de falsos positivos en la detección de malaria observado en modelos previos de reconocimiento automático.
Muscle dysfunction and exercised-based rehabilitation in cardiac diseases: From muscular physiology in animals to prognosis impact on patients
(Universitat de Barcelona, 2023-01-24) Cabrera-Aguilera, Ignacio Alfredo; Farré López, Núria; Universitat de Barcelona. Departament de Biomedicina
[eng] Cardiovascular diseases (CVD) such heart failure (HF) or acute coronary syndrome (ACS) impairs muscular dysfunction that affect cardiorespiratory fitness. Animals models realistically mimicking CVD, including the well-known diaphragm dysfunction observed in HF patients. The widely used isoproterenol-induced HF model has not been explored for new insights to improve muscular dysfunction. From a clinical perspective, the impact of muscular dysfunction is multiple but preventable and reversible. Exercise-based cardiac rehabilitation programs (EB-CRP) have a recommendation class 1A in clinical guidelines, but remains underused. Participant initially are evaluated and stratified with routine protocols to refer the best setting and conditions for exercise training (ET). Despite benefits of exercise training and prognosis studies, remains unknown the prognosis of these patients who accept all the others components of cardiac rehabilitation but not performing training. In the same way, it is unknown if these tools have a prognosis utility. The aim of this thesis was, by one hand, to describe diaphragmatic contractility of an animal model never described before. On the other hand, was to compare clinical outcomes and asses the predictive value of risk stratification (RS) between patients with different compliance and initial attitude for accept or reject exercise component of cardiac rehabilitation. Using ex-vivo and in-vivo measure of muscular function, conventional isoproterenol-induced HF model increases diaphragm contraction, a finding contrary to what is observed in patients with HF. In EB-CRP, completion of ET after ACS was associated with improved prognosis and with reclassification to low-risk. Only half of the patients completed the ET program and finally a Spanish easy-to-calculate risk score offers unreported robust prognostic information. No-exercise groups were independently associated with the worst outcomes. EB-CRP with participation in exercise component changed risk stratification, improving classification and prognosis.
Depletion of aneuploid cells in epithelial tissues is shaped by cell-to-cell interactions
(Universitat de Barcelona, 2025-04-04) Fusari, Elena; Milán Kalbfleisch, Marco; Universitat de Barcelona. Departament de Biomedicina
[eng] Aneuploidy, the major cause of miscarriages, is pervasive in early human embryos, and later in life, it correlates with pathological conditions including cancer and other ageing-related conditions. At the cellular level, both gains and losses of chromosome are deleterious and result in growth defects. At the organismal level, almost all trisomies and monosomies are lethal and those that are compatible with life are associated with severe developmental defects. Surprisingly, 80% of blastocysts are reported to be aneuploid mosaics. In disease, aneuploidy is present in 90% of human solid tumors, it confers selective advantage to cancer cells and significantly contributes to tumorigenesis. Identification of the mechanisms underlying the elimination of aneuploid cells is therefore relevant in development and disease. Since aneuploid cells in vivo generally emerge as a consequence of missegregation events during cell division, it is often found in mosaics. One mechanism that has been proposed to participate in the recognition and elimination of aneuploid cells to ensure correct development and tissue homeostasis in cell competition, a process were difference in fitness are sensed and less fit cells are actively eliminated by fitter cells. In order to study aneuploidy it is crucial to dispose of good experimental models. In particular, sequence-specific methods allow to differentiate between general and karyotype-specific effects of gene dosage imbalance. Unfortunately, such strategies have been developed mainly in vitro therefore lacking the ability to characterize the impact of the interaction between aneuploid and wild type cells. Here, we developed a strategy based on the Flp/FRT sequence-specific recombination system to generate labelled segmental aneuploid cells within epithelial tissues of Drosophila. We generated cells carrying molecularly defined segmental monosomies and trisomies and characterized their immediate impact on cellular behavior, growth and survival. Our data reveal signs of out-competition of cells carrying monosomies in genomic regions devoid of previously known haploinsufficient genes due to newly identified haploinsufficient genes or to cumulative haploinsufficiency. Notably, these mechanisms of cell competition rely on distinct molecular pathways, namely Xrp1-mTor-dependent or -independent cell competition. By simultaneously inducing cells carrying monosomies and trisomies of the same genomic location, we present evidence that segmental trisomies potentiate or alleviate the negative effects of the monosomy on growth. We describe a case of supercompetition of the trisomies, that overgrows respect to control cells at the expenses of the monosomic cells, and a case of growth compensation, where trisomic cells induce compensatory proliferation of otherwise outcompeted monosmies. Furthermore, we describe two triplosensitive regions. Overall, our results reveal that the genome is full of dosage-sensitive loci and uncover a key role of cell interactions and specifically of cell competition in defining the in vivo elimination of aneuploid cells.
Implications of astrocytic RTP801 in neuroinflammation and neurodegeneration in Alzheimer’s disease
(Universitat de Barcelona, 2024-11-29) Chicote González, Almudena; Malagelada Grau, Cristina; Universitat de Barcelona. Departament de Biomedicina
[eng] The central nervous system (CNS), consisting of the brain and spinal cord, is supported by various cells including neurons, glial cells, and blood vessel cells. Neurons are responsible for transmitting signals, while astrocytes, once considered merely support cells, are now recognized as vital for CNS function. Astrocytes maintain CNS balance, assist in brain defense, and regulate several processes. Historically, astrocytes were 7irst identi7ied in the mid-1800s and develop from radial glia, dividing and transforming into mature astrocytes during brain development. They continue to proliferate in the adult brain but at a low rate. Astrocytes are critical in maintaining CNS homeostasis, including regulating ions, water, and neurotransmitter levels. They also play a key role in energy supply to neurons by converting glucose into lactate. Moreover, astrocytes are essential for synaptic regulation, promoting synapse formation and stability, and maintaining the blood-brain barrier. Astrocytes also contribute to neurovascular coupling and waste clearance via the glymphatic system, and they are involved in regulating blood 7low and oxygen levels. In neuroin7lammatory and neurodegenerative diseases, astrocytes can become reactive, leading to dysfunction. In diseases like Alzheimer's and Parkinson's, astrocytes lose their ability to support neurons and remove toxic substances, contributing to disease progression. Astrocyte reactivity is in7luenced by various signaling pathways, but the mechanisms of their dysregulation remain unclear. Alzheimer’s disease is the most common form of dementia worldwide, affecting millions of people. It is both sporadic (1%) and hereditary, with age being the higher risk factor. Inherited Alzheimer’s disease is caused by mutations in the APP, PSEN1 or PSEN 2 genes. Having the APOE ε4 gene is the primary genetic risk factor for late-onset Alzheimer’s disease. The disease is characterized by cortical atrophy and enlargement of the ventricles. A central feature of AD is the accumulation of amyloid-β (Aβ) peptides, derived from amyloid precursor protein, prone to forming plaques. These plaques are linked to cognitive decline, particularly in their oligomeric form. Another hallmark of AD is the formation of neuro7ibrillary tangles, caused by hyperphosphorylation of tau proteins, which disrupt neuron function and lead to cell death. Neuroin7lammation also plays a key role, with microglia and astrocytes contributing to both Aβ clearance and neurodegeneration through in7lammatory pathways like the NLRP3 in7lammasome, exacerbating synaptic loss and further progression of the disease. AD treatments are divided into symptomatic and disease-modifying categories, although no cure exists yet. Promising new diagnostic biomarkers and therapies focusing on prevention and early intervention represent the future direction of AD management. RTP801, is a stress-induced protein involved in regulating various cellular processes like metabolism, oxidative stress, autophagy, and cell fate. It plays a role in in7lammatory, metabolic, neurodegenerative diseases, and cancer. Its expression is upregulated by stressors like hypoxia, DNA damage, and metabolic imbalances. RTP801 is primarily located in the cytoplasm but also in mitochondria, cell membranes, and the nucleus. A key function of RTP801 is inhibiting the mTOR pathway, which regulates cellular growth and autophagy. Elevated levels of RTP801 are linked to neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's, where it suppresses mTOR activity, leading to neuronal damage. Its inhibition in disease models has shown potential therapeutic bene7its by preventing cognitive decline and reducing in7lammation. This thesis aims to explore astrocytic RTP801's contribution to neurodegeneration and neuroin7lammation in AD using the 5xFAD mouse model and a novel triculture in vitro model. The goals include determining its effects on cognitive de7icits, neuron morphology, functional connectivity, neuroin7lammatory pathways, and intercellular crosstalk, as well as assessing its role in Aβ clearance.
Exploring extracellular small RNAs as potential early biomarkers and mediators in the pathogenesis of Huntington’s disease
(Universitat de Barcelona, 2024-10-18) Herrero Lorenzo, Marina; Martí Puig, Eulàlia; Gámez Valero, Ana; Universitat de Barcelona. Departament de Biomedicina
[eng] Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of CAG triplets in the huntingtin gene (HTT). The main neuropathological signs of HD include the presence of cytoplasmic aggregates of the protein produced by mutated HTT (mHTT), massive loss of medium spiny neurons in the striatum, and cortical degeneration. In current clinical practice, symptomatic patients are diagnosed based on the appearance of a complex constellation of clinical symptoms, including progressive motor abnormalities, neuropsychiatric disorders, early cognitive impairment, and dementia, among others. However, genetic analysis allows the use of predictive tests to identify carriers of the genetic mutation in presymptomatic phases, who may present progressive brain changes and alterations in cognitive performance long before the diagnosis of the disease characterized by the appearance of motor symptoms. Current treatments for HD only provide symptomatic relief, and the most recent results from clinical trials investigating gene therapies have not shown significant efficacy so far. Combining predictive genetic testing with novel molecular biomarkers at early stages could help improve clinical trial design for HD, by selecting the most appropriate patients, stratification of patients for interventions, monitoring response to treatment, and improving the efficiency of new clinical trials.
Nous abordatges en l'estudi de les malalties autoimmunitàries
(Universitat de Barcelona, 2024-10-30) Pérez Isidro, Albert; Ruiz Ortiz de Arizabaleta, Estíbaliz; Espinosa Garriga, Gerard; Universitat de Barcelona. Departament de Biomedicina
[cat] HIPÒTESIS: Un augment del nombre de biomarcadors disponibles amplia la informació a l’abast del clínic i ajuda a una millor la presa de decisions mèdiques i terapèutiques per a l’atenció dels pacients amb malalties autoimmunitàries. La quantificació dels limfòcits B productors d’autoanticossos anti- dsDNA en sang perifèrica aporta informació complementària a la qual s’obté habitualment amb la quantificació d’autoanticossos anti-dsDNA en sèrum, per a l’avaluació de l’activitat del lupus eritematós sistèmic i del risc de desenvolupar nefritis lúpica. La determinació de l’isotip IgA dels autoanticossos inclosos en els criteris classificatoris de l’esclerosi sistèmica, a més dels anti-Ro52, redueix la proporció de pacients seronegatius i millora l’estratificació dels pacients en funció de les manifestacions clíniques. La determinació dels isotips IgM i IgA dels autoanticossos inclosos en els criteris classificatoris de l’artritis reumatoide, a més d’altres biomarcadors associats a la malaltia (RA33, CEP-1 i KL-6), redueix la proporció de pacients seronegatius i millora l’estratificació del risc de patir malaltia pulmonar intersticial difusa. A més, la determinació d’aquests biomarcadors s’associa a la mortalitat en un seguiment a quatre anys. OBJECTIUS: 1. ESTUDI 1: Quantificar els limfòcits B productors d’autoanticossos anti-dsDNA en pacients amb Lupus Eritematós Sistèmic (LES) (Publicació 1). 1.1. Estandarditzar un mètode ELISpot per a quantificar els limfòcits B productors d’autoanticossos anti-dsDNA en sang perifèrica. 1.2. Comparar els resultats de l’ELISpot amb la quantificació d’autoanticossos anti-dsDNA i l’estudi del complement en sèrum per a l’avaluació de l’activitat del LES. 1.3. Correlacionar els resultats de l’ELISpot amb les manifestacions clíniques de la malaltia. 1.4. Avaluar la capacitat predictora dels resultats de l’ELISpot per a l’aparició de brots de LES. 2. ESTUDI 2: Augmentar el nombre de biomarcadors disponibles per al diagnòstic i l’estratificació dels pacients amb Esclerosi Sistèmica (ES) (Publicació 2). 2.1. Determinar la prevalença dels autoanticossos ACA, ATA, RP3 (inclosos en els criteris classificatoris de l’ES) i els anti-Ro52 d’isotip IgA i IgG. 2.2. Correlacionar la presència dels diferents isotips d’autoanticossos amb les diferents manifestacions clíniques de l’ES. 3. ESTUDI 3: Augmentar el nombre de biomarcadors disponibles per al diagnòstic i l’avaluació pronòstica de l’Artritis Reumatoide (AR) (Publicació 3). 3.1. Determinar la prevalença dels autoanticossos ACPA, FR (inclosos en els criteris), RA33 d’isotip IgM, IgA i IgG i CEP-1 d’isotip IgG i IgA. 3.2. Determinar la prevalença de la molècula soluble KL-6. 3.3. Correlacionar la presència d’aquests biomarcadors (autoanticossos i KL-6) amb la malaltia pulmonar intersticial difusa associada a l’AR. 3.4. Avaluar els factors de risc associats a la mortalitat en un seguiment de quatre anys.

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