Illuminating cAMP signalling in neurodegenerative diseases: from synaptic plasticity mechanisms to therapeutic opportunities
[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 improve...
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| Tipo de recurso: | tesis doctoral |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/224734 |
| Acceso en línea: | https://hdl.handle.net/2445/224734 http://hdl.handle.net/10803/695809 |
| Access Level: | acceso embargado |
| Palabra clave: | Neurociències Neuropatologia Neuroplasticitat Corea de Huntington Malaltia d'Alzheimer Neurosciences Enfermedades del sistema nervioso Neuroplasticity Huntington's chorea Alzheimer's disease |
| Sumario: | [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. |
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