Tissue Clearing and Expansion Methods for Imaging Brain Pathology in Neurodegeneration : From Circuits to Synapses and Beyond

Studying the structural alterations occurring during diseases of the nervous system requires imaging heterogeneous cell populations at the circuit, cellular and subcellular levels. Recent advancements in brain tissue clearing and expansion methods allow unprecedented detailed imaging of the nervous system through its entire scale, from circuits to synapses, including neurovascular and brain lymphatics elements. Here, we review the state-of-the-art of brain tissue clearing and expansion methods, mentioning their main advantages and limitations, and suggest their parallel implementation for circuits-to-synapses brain imaging using conventional (diffraction-limited) light microscopy -such as confocal, two-photon and light-sheet microscopy- to interrogate the cellular and molecular basis of neurodegenerative diseases. We discuss recent studies in which clearing and expansion methods have been successfully applied to study neuropathological processes in mouse models and postmortem human brain tissue. Volumetric imaging of cleared intact mouse brains and large human brain samples has allowed unbiased assessment of neuropathological hallmarks. In contrast, nanoscale imaging of expanded cells and brain tissue has been used to study the effect of protein aggregates on specific subcellular structures. Therefore, these approaches can be readily applied to study a wide range of brain processes and pathological mechanisms with cellular and subcellular resolution in a time- and cost-efficient manner. We consider that a broader implementation of these technologies is necessary to reveal the full landscape of cellular and molecular mechanisms underlying neurodegenerative diseases

Gene expression parallels synaptic excitability and plasticity changes in Alzheimer's disease

Altres ajuts: CIBERNED CB06/05/0042 i BrightFocus Foundation (A2014417S)Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal accumulation of β-amyloid and tau and synapse dysfunction in memory-related neural circuits. Pathological and functional changes in the medial temporal lobe, a region essential for explicit memory encoding, contribute to cognitive decline in AD. Surprisingly, functional imaging studies show increased activity of the hippocampus and associated cortical regions during memory tasks in presymptomatic and early AD stages, whereas brain activity declines as the disease progresses. These findings suggest an emerging scenario where early pathogenic events might increase neuronal excitability leading to enhanced brain activity before clinical manifestations of the disease, a stage that is followed by decreased brain activity as neurodegeneration progresses. The mechanisms linking pathology with synaptic excitability and plasticity changes leading to memory loss in AD remain largely unclear. Recent studies suggest that increased brain activity parallels enhanced expression of genes involved in synaptic transmission and plasticity in preclinical stages, whereas expression of synaptic and activity-dependent genes are reduced by the onset of pathological and cognitive symptoms. Here, we review recent evidences indicating a relationship between transcriptional deregulation of synaptic genes and neuronal activity and memory loss in AD and mouse models. These findings provide the basis for potential clinical applications of memory-related transcriptional programs and their regulatory mechanisms as novel biomarkers and therapeutic targets to restore brain function in AD and other cognitive disorders

Revealing cell vulnerability in Alzheimer's disease by single-cell transcriptomics

Acord transformatiu CRUE-CSICAlzheimer's disease (AD) is a neurodegenerative disorder that by affecting specific brain cell types and regions cause severe pathological and functional changes in memory neural circuits. A comprehensive knowledge of the pathogenic mechanisms underlying AD requires a deeper understanding of the cell-specific pathological responses through integrative molecular analyses. Recent application of high-throughput single-cell transcriptomics to postmortem tissue has proved powerful to unravel cell susceptibility and biological networks responding to amyloid and tau pathologies. Here, we review single-cell transcriptomic studies successfully applied to decipher cell-specific gene expression programs and pathways in the brain of AD patients. Transcriptional information reveals both specific and common gene signatures affecting the major cerebral cell types, including astrocytes, endothelial cells, microglia, neurons, and oligodendrocytes. Cell type-specific transcriptomes associated with AD pathology and clinical symptoms are related to common biological networks affecting, among others pathways, synaptic function, inflammation, proteostasis, cell death, oxidative stress, and myelination. The general picture that emerges from systems-level single-cell transcriptomics is a spatiotemporal pattern of cell diversity and biological pathways, and novel cell subpopulations affected in AD brain. We argue that broader implementation of cell transcriptomics in larger AD human cohorts using standardized protocols is fundamental for reliable assessment of temporal and regional cell-type gene profiling. The possibility of applying this methodology for personalized medicine in clinics is still challenging but opens new roads for future diagnosis and treatment in dementia

Crtc1 activates a transcriptional program deregulated at early Alzheimer's disease-related stages

Cognitive decline is associated with gene expression changes in the brain, but the transcriptional mechanisms underlying memory impairments in cognitive disorders, such as Alzheimer's disease (AD), are largely unknown. Here, we aimed to elucidate relevant mechanisms responsible for transcriptional changes underlying early memory loss in AD by examining pathological, behavioral, and transcriptomic changes in control and mutant β-amyloid precursor protein(APPSw,Ind) transgenic mice during aging. Genome-wide transcriptome analysis using mouse microarrays revealed deregulation of a gene network related with neurotransmission, synaptic plasticity, and learning/memory in the hippocampus of APPSw,Ind mice after spatial memory training. Specifically, APPSw,Ind mice show changes on a cAMP-responsive element binding protein(CREB)- regulated transcriptional program dependent on the CREB-regulated transcription coactivator-1 (Crtc1). Interestingly, synaptic activity and spatial memory induces Crtc1 dephosphorylation (Ser151), nuclear translocation, and Crtc1-dependent transcription in the hippocampus, and these events are impaired in APPSw,Ind mice at early pathological and cognitive decline stages. CRTC1-dependent genes and CRTC1 levels are reduced in human hippocampus at intermediate Braak III/IV pathological stages. Importantly, adeno-associated viral-mediated Crtc1 overexpression in the hippocampus efficiently reverses Aβ-induced spatial learning and memory deficits by restoring a specific subset of Crtc1 target genes. Our results reveal a critical role of Crtc1-dependent transcription on spatial memory formation and provide the first evidence that targeting brain transcriptome reverses memory loss in AD

CRTC1 function during memory encoding is disrupted in neurodegeneration

Altres ajuts: Alzheimer's disease research program of the BrightFocus Foundation (Ref. A2014417)Methods: We evaluated the activation of CRTC1 in the hippocampus of control mice and mice lacking the Alzheimer's disease-linked presenilin genes (presenilin conditional double knockout [PS cDKO]) after one-trial contextual fear conditioning by using biochemical, immunohistochemical, and gene expression analyses. PS cDKO mice display classical features of neurodegeneration occurring in Alzheimer's disease including age-dependent cortical atrophy, neuron loss, dendritic degeneration, and memory deficits.Results: Context-associative learning, but not single context or unconditioned stimuli, induces rapid dephosphorylation (Ser151) and translocation of CRTC1 from the cytosol/dendrites to the nucleus of hippocampal neurons in the mouse brain. Accordingly, context-associative learning induces differential CRTC1-dependent transcription of c-fos and the nuclear receptor subfamily 4 (Nr4a) genes Nr4a1-3 in the hippocampus through a mechanism that involves CRTC1 recruitment to CRE promoters. Deregulation of CRTC1 dephosphorylation, nuclear translocation, and transcriptional function are associated with long-term contextual memory deficits in PS cDKO mice. Importantly, CRTC1 gene therapy in the hippocampus ameliorates context memory and transcriptional deficits and dendritic degeneration despite ongoing cortical degeneration in this neurodegeneration mouse model. Conclusions: These findings reveal a critical role of CRTC1 in the hippocampus during associative memory, and provide evidence that CRTC1 deregulation underlies memory deficits during neurodegeneration

Short-Term Environmental Enrichment Rescues Adult Neurogenesis and Memory Deficits in APPSw,Ind Transgenic Mice

Epidemiological studies indicate that intellectual activity prevents or delays the onset of Alzheimer's disease (AD). Similarly, cognitive stimulation using environmental enrichment (EE), which increases adult neurogenesis and functional integration of newborn neurons into neural circuits of the hippocampus, protects against memory decline in transgenic mouse models of AD, but the mechanisms involved are poorly understood. To study the therapeutic benefits of cognitive stimulation in AD we examined the effects of EE in hippocampal neurogenesis and memory in a transgenic mouse model of AD expressing the human mutant β-amyloid (Aβ) precursor protein (APPSw,Ind). By using molecular markers of new generated neurons (bromodeoxiuridine, NeuN and doublecortin), we found reduced neurogenesis and decreased dendritic length and projections of doublecortin-expressing cells of the dentate gyrus in young APPSw,Ind transgenic mice. Moreover, we detected a lower number of mature neurons (NeuN positive) in the granular cell layer and a reduced volume of the dentate gyrus that could be due to a sustained decrease in the incorporation of new generated neurons. We found that short-term EE for 7 weeks efficiently ameliorates early hippocampal-dependent spatial learning and memory deficits in APPSw,Ind transgenic mice. The cognitive benefits of enrichment in APPSw,Ind transgenic mice were associated with increased number, dendritic length and projections to the CA3 region of the most mature adult newborn neurons. By contrast, Aβ levels and the total number of neurons in the dentate gyrus were unchanged by EE in APPSw,Ind mice. These results suggest that promoting the survival and maturation of adult generated newborn neurons in the hippocampus may contribute to cognitive benefits in AD mouse models

Role of CREB/CRTC1-regulated gene transcription during hippocampal-dependent memory in Alzheimer’s disease mouse models

Cambios en la transcripción de genes en el cerebro están asociados a alteraciones sinápticas y deterioro cognitivo tanto durante el envejecimiento normal como en trastornos cognitivos relacionados a la edad. Sin embargo, poco se sabe acerca de los mecanismos moleculares que subyacen alteraciones de la expresión génica durante la enfermedad de Alzheimer. El factor de transcripción CREB regula la expresión de genes esenciales para los mecanismos de plasticidad sináptica a largo plazo y consolidación de la memoria. La transcripción de genes dependientes de CREB está regulada por coactivadores transcripcionales como CBP, p300 y la familia de coactivadores CRTC. CRTC1 es la isoforma más abundante en neuronas y se ha implicado en la regulación del crecimiento dendrítico, la plasticidad sináptica a largo plazo y la memoria. Las funciones específicas de CRTC1 sobre las alteraciones transcripcionales, disfunción sináptica y pérdida de memoria durante la enfermedad de Alzheimer son desconocidas. En esta tesis doctoral se ha investigado el papel de la transcripción regulada por CREB/CRTC1 durante procesos de memoria dependiente del hipocampo en dos modelos murinos bien establecidos de patología amiloide y neurodegeneración: 1) el modelo de ratón transgénico APPsw,Ind que expresa el gen APP humano con las mutaciones sueca e Indiana asociadas a la enfermedad de Alzheimer de tipo hereditario, y 2) un ratón doble knockout condicional (PS cDKO) que carece de los genes de la presenilina (PS1 y PS2) en neuronas excitatorias del cerebro adulto. Hemos observado que la inducción de actividad neuronal así como el entrenamiento en paradigmas de memoria espacial y asociativa induce la defosforilación de CRTC1 en Ser151, lo que promueve la translocación de CRTC1 al núcleo de neuronas hipocampales de ratones adultos. Curiosamente, durante estadíos patológicos iniciales ambos modelos muestran una disminución significativa en la expresión de genes diana de CREB/CRTC1 durante tareas de memoria dependiente de hipocampo. Cabe destacar que la desfosforilación en Ser151 y la translocación nuclear de CRTC1 se ven alteradas en las neuronas del hipocampo en ambos modelos, coincidiendo con déficits iniciales de memoria dependiente de hipocampo. Estos resultados indican que la función de CRTC1 se ve comprometida en ambos modelos de ratones, lo que sugiere que la transcripción dependiente de CRTC1 puede verse afectada por diferentes vías patogénicas. Es importante destacar que también hemos observado alteración en la expresión génica regulada por CRTC1 en muestras de hipocampo humano con patología de enfermedad de Alzheimer. Además, hemos determinado que la sobreexpresión de CRTC1 en el hipocampo mejora los déficits de transcripción y de memoria en etapas patológicas tempranas en ambos modelos experimentales, lo que sugiere que CRTC1 puede ser una valiosa diana terapéutica para la enfermedad de Alzheimer y otras demencias neurodegenerativas. En resumen, estos resultados apoyan un modelo en el que distintos mecanismos patogénicos comprometen la transcripción regulada por CRTC1 necesaria para la plasticidad sináptica y la memoria, lo que contribuyendo a la neurodegeneración y demencia durante la enfermedad de Alzheimer. Estos resultados revelan un papel crítico de la señalización de CRTC1 en el hipocampo durante memoria espacial y asociativa, y sugieren que la modulación de CRTC1 puede ser una importante estrategia terapéutica para la enfermedad de Alzheimer y trastornos neurodegenerativos relacionados.Altered gene transcription in the brain is associated with impaired synaptic function and cognitive decline during normal aging and age-related cognitive disorders. However, the molecular mechanisms underlying deregulation of gene expression during Alzheimer’s disease remain largely unknown. Activity-dependent gene expression regulated by the transcription factor cAMP-response element binding protein (CREB) is essential for long-term synaptic plasticity and memory consolidation. CREB-dependent transcription is regulated by transcriptional coactivators including CREB binding protein (CBP), p300 and the CREB-regulated transcription coactivators (CRTCs). CRTC1 is the most abundant isoform in neurons and has been implicated in the regulation of dendritic growth, long-term synaptic plasticity and memory. Importantly, the specific roles of CRTC1 on transcriptional changes, synaptic dysfunction and memory loss during Alzheimer´s disease-related pathology are unknown. In this doctoral thesis I have investigated the role of CREB/CRTC1-regulated transcription during hippocampal-dependent memory in two well-established mouse models of amyloid pathology and neurodegeneration: 1) the APPSw,Ind transgenic mouse model of amyloidosis expressing the human APP gene harboring the familial-AD linked Swedish and Indiana mutations, and 2) a Presenilin conditional double knockout (PS cDKO) mouse model lacking the presenilin genes (PS1 and PS2) in forebrain neurons of the adult brain. We found that induction of neuronal activity as well as spatial and associative memory training induce CRTC1 dephosphorylation at Ser151, which leads to a time-dependent CRTC1 translocation to the nucleus in hippocampal neurons of the adult mouse brain. Interestingly, during early pathological stages both APPSw,Ind and PS cDKO mice show significant decreased expression of CRTC1-dependent CREB target genes induced by hippocampal-dependent memory tasks. Notably, CRTC1 dephosphorylation at Ser151 and nuclear translocation are altered in hippocampal neurons of both mouse models coinciding with early hippocampal-dependent memory deficits. These results indicate that CRTC1 function is compromised in both mouse models, suggesting that CRTC1-dependent transcription may be affected by different pathogenic pathways. Importantly, we provide evidence that CRTC1-regulated transcription is also altered in the human hippocampus during Alzheimer’s disease pathology. Furthermore, we show that increasing CRTC1 function in the hippocampus ameliorates transcriptional and memory deficits at early pathological stages in both mouse models, suggesting that targeting CRTC1 signaling may be a valuable therapeutic strategy for Alzheimer’s disease and related neurodegenerative dementias. Taken together, our results support a model in which distinct pathogenic mechanisms compromise CREB/CRTC1-dependent transcription required for synaptic plasticity and memory, which contributes to neurodegeneration and dementia during Alzheimer’s disease-related pathology. These findings reveal a critical role of CRTC1 signaling in the hippocampus during spatial and contextual associative memory, and provide evidence that targeting CRTC1 signaling may be a valuable therapeutic strategy for Alzheimer’s disease and related neurodegenerative disorders

Is phosphorylated tau a good biomarker of synapse pathology in Alzheimer's disease ?

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