Transcriptome meta-analysis reveals a central role for sex steroids in the degeneration of hippocampal neurons in Alzheimer’s disease

BACKGROUND: Alzheimer’s disease is the most prevalent form of dementia. While a number of transcriptomic studies have been performed on the brains of Alzheimer’s specimens, no clear picture has emerged on the basis of neuronal transcriptional alterations linked to the disease. Therefore we performed a meta-analysis of studies comparing hippocampal neurons in Alzheimer’s disease to controls. RESULTS: Homeostatic processes, encompassing control of gene expression, apoptosis, and protein synthesis, were identified as disrupted during Alzheimer’s disease. Focusing on the genes carrying out these functions, a protein-protein interaction network was produced for graph theory and cluster exploration. This approach identified the androgen and estrogen receptors as key components and regulators of the disrupted homeostatic processes. CONCLUSIONS: Our systems biology approach was able to identify the importance of the androgen and estrogen receptors in not only homeostatic cellular processes but also the role of other highly central genes in Alzheimer’s neuronal dysfunction. This is important due to the controversies and current work concerning hormone replacement therapy in postmenopausal women, and possibly men, as preventative approaches to ward off this neurodegenerative disorder

Revisión bibliográfica de los vínculos genéticos, bioquímicos y moleculares de la enfermedad de Alzheimer

Introducción: la enfermedad de Alzheimer (EA) es la causa más frecuente de demencia en Occidente. Se caracteriza por presentar un inicio insidioso y progresión lenta, con una evolución media de unos 8 o 10 años desde el inicio hasta la muerte. La edad de inicio de los síntomas de la EA está en torno a los 65 años, duplicando la prevalencia cada 5 años. Como objetivo general de esta revisión bibliográfica de la literatura, se pretende identificar los biomarcadores asociados a la EA. Como objetivos específicos, tratar en profundidad el papel de la apolipo-proteína E en el desarrollo tardío de esta enfermedad, en concreto la presencia del alelo E4 en la misma, que determina una alteración del perfil lipídico y como consecuencia una ateroesclerosis temprana; identificar otros factores de riesgo; así como otros factores protectores de la enfermedad, relacionados sobre todo con el estilo de vida. Metodología: se realizó una revisión de la literatura científica existente desde 1998 hasta 2017, en bases de datos y buscadores biomédicos como Pubmed, entre otros. Para ello, se utilizó el método PICO, basado en la evidencia científica, siguiendo unos criterios de inclusión y exclusión predeterminados. Resultados: las lesiones histológicas típicas de la EA son los depósitos intracelulares de tau hiperfosforilada y las placas de beta amiloide, cuyo componente principal es el péptido beta-amiloide de 42 aminoácidos (βA42). Los pacientes con EA presentan una concentración disminuida de beta-amiloide 42 y un incremento de los valores de tau total y tau fosforilada en el líquido cefalorraquídeo. Los factores de riesgo asociados a la enfermedad esporádica son la edad; el sexo; los factores de riesgo cardiovascular: diabetes, hipertensión arterial, dislipemia, obesidad, tabaquismo; y la depresión mayor. Como factores protectores se han descrito: un alto nivel cultural; dieta mediterránea; prevención de los factores de riesgo cardiovascular; y el ejercicio físico regular. Conclusiones: es preciso intervenir de manera agresiva y temprana sobre la dislipemia en los pacientes diagnosticados de la mutación en el alelo 4 de la APOE, que presumiblemente desarrollarán en la ancianidad la EA; y por otro lado, llevar a cabo las intervenciones de prevención primaria y secundaria en la población general adulta de los factores de riesgo cardiovascular para evitar futuros eventos cardio y cerebrovasculares, así como el deterioro cognitivo.<br /

Analysis of the expression of taste and olfactory receptors in choroid plexus and orbitofrontal cortex of Alzheimer’s disease patients

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive memory loss and cognitive deterioration, attributed to neuropathological lesions within specific regions in the brain. However, other areas of the brain such as the choroid plexus and the orbitofrontal cortex, have gained much attention since the choroid plexus is a multifunctional tissue responsible for a wide range of functions crucial to the central nervous system and the orbitofrontal cortex is considered among the most polymodal regions of the brain. Previous studies have shown the expression of taste and olfactory transduction pathways in rat choroid plexus as well as taste and olfactory regulation and activation of orbitofrontal cortex. Therefore the present work aimed to study the expression of taste and olfactory receptors in the choroid plexus and orbitofrontal cortex of AD patients. Towards this aim, transcriptomic analysis of these chemoreceptors were performed by means of Real-time quantitative Polymerase Chain Reaction (RT-qPCR), and compared between AD patients (at different Braak stages of the disease) and age-matched controls. Transcriptomic analysis indicated that orbitofrontal cortical olfactory receptors (ORs) and taste receptors (TASRs) are expressed and regulated at different stages of AD in female patients, whereas in male this regulation was not observed. Moreover, olfactory receptors OR2K2, OR2H2, and OR1L8 and taste receptor TAS2R14 were downregulated at Braak stages I, V, VI, compared to age-matched controls in the orbitofrontal cortex of female AD patients. The strongest differences were found at Braak stage I of AD, suggesting that dysregulation of ORs and TASRs is an early event in the pathogenesis of AD in female patients. Also, that ORs and TASRs in the orbitofrontal cortex might be differently affected in male and female AD patients. These receptors we also found to be expressed in choroid plexus, however, due to their low expression we were not able to quantify the relative mRNA levels by RT-qPCR. Nevertheless further studies are needed in order to strengthen these findings and to elucidate their potential physiologic functions in this brain area.A doença de Alzheimer é uma doença neurodegenerativa crónica caracterizada por uma perda progressiva da memória e défice das capacidades cognitivas. Estes défices cognitivos são atribuídos a lesões neuropatológicas intrínsecas a regiões específicas do cérebro. No entanto, outras áreas do cérebro, como o plexo coroide e o córtex orbito-frontal, têm sido objeto de grande atenção uma vez que o plexo coroide é um tecido multifuncional responsável por uma vasta gama de funções cruciais ao sistema nervoso central e o córtex orbito-frontal é considerado uma das regiões mais polimodais do cérebro. Estudos anteriores demonstraram a expressão das vias de transdução do gosto e do olfacto no plexo coroide de ratos, bem como a ativação e regulação olfativa e gustativa do córtex orbito-frontal. Inclusivamente, num estudo anterior foi demonstrado que no plexo coroide de ratos a expressão de genes relacionados com o sabor e o olfato se encontram sob o controlo de hormonas esteróides sexuais. Por esse motivo, o presente trabalho teve como objetivo estudar a expressão dos recetores olfativos e gustativos no plexo coroide e no córtex orbito-frontal de pacientes com doença de Alzheimer de ambos os sexos. A fim de atingir esse objetivo, foi necessário desenhar primers específicos para esses recetores e optimizar as várias condições para obter curvas de calibração eficientes para a análise transcritómica quantitativa. A análise do transcriptoma destes quimiorrecetores foi então efectuada através de reação em cadeia da polimerase quantitativa em tempo real (em inglês, Real-time quantitative Polymerase Chain Reaction), e os níveis de expressão foram comparados entre pacientes com doença de Alzheimer (em diferentes estágios da doença) e controlos pareados. A análise do transcriptoma revelou que os recetores olfativos (ORs) e gustativos (TASRs) orbito-frontal corticais se expressam e se encontram regulados em alguns estágios da doença em pacientes do sexo feminino, enquanto que em pacientes do sexo oposto essa mesma regulação nao foi observada. Nomeadamente, a expressão dos recetores olfativos OR2K2, OR2H2 e OR1L8 e o recetor gustativo TAS2R14 encontra-se regulada negativamente nos estágios de Braak I, V e VI em comparação com os controlos pareados em córtex orbito-frontal de pacientes do sexo feminino com doença de Alzheimer. As diferenças mais evidentes foram encontradas no estágio de Braak I, sugerindo que a desregulação da expressão dos recetores olfactivos e gustativos é um evento que ocorre precocemente na patologia de Alzheimer em pacientes do sexo feminino. Apesar da sua tendência para aumentar em estágios mais avançados, possivelmente em resposta à progressão da doença (ao nível bioquímico e estrutural), o córtex orbito-frontal parece não ser capaz de restaurar os níveis observados em pacientes controlo. Além disso, estes resultados sugerem também que a expressão dos ORs e os TASRs no córtex orbito-frontal pode ser afetada de forma distinta entre pacientes de Alzheimer de um ou outro sexo. Inclusivamente, verificou-se que estes recetores também se expressam em plexo coroide, no entanto, devido à sua baixa expressão não foi possível quantificar os seus níveis relativos de mRNA por PCR quantitativa em tempo real. A utilização de tecidos post-mortem humanos para estudos de expressão génica é particularmente desafiador. Além do problema do RNA danificado, temos de enfrentar um elevadíssimo grau de variabilidade biológica dentro de um conjunto de amostras. As variações dos parâmetros individuais como a idade, a massa corporal, a saúde, mas também a causa e circunstâncias da morte e o intervalo post-mortem podem conduzir a um conjunto de amostras bastante inomogéneo. Adicionalmente, mais estudos são necessários a fim de reforçar estas conclusões e elucidar as possíveis funções fisiológicas destes quimiorrecetores nestas áreas do cérebro

The Effects of Induced Neural Stem Cell Plasticity and Neurogenesis on the Pathology of Alzheimer's Disease

Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder that leads to a steady deterioration in mental, behavioral, and social abilities, impairing a person's capacity for independent functioning. It is the most prevalent form of neurodegenerative disorder and is currently the most common cause of dementia. According to the classical paradigm of AD's pathogenesis, neurotoxic protein (amyloid beta-42 peptides and hyperphosphorylated tau) gradually aggregates in the brain over several years, causing neuronal dysfunction, synaptic degeneration, neuronal death, and shrinkage of the brain in the process, which are also a few of the hallmarks of AD. As currently there is no cure for AD, it is marked as one of the leading causes of death among the elderly population. For several decades, the primary goal of AD research has been to avoid neuronal death and synaptic failure. Prevention of accumulation of amyloid plaques and tau-neurofibrillary tangles, synaptic degeneration, and reduction in the neurotransmitter acetylcholine was prioritized as therapy options for AD. However, contemporary therapies cause a meager slowdown in the cognitive decline of AD patients and these medications are far from curing the disease. Perhaps a prominent alternative for treating AD patients would emerge when we better understand the disease pathogenesis. Although the exact cause of AD is still unknown, recent studies suggest a multidimensional causality between numerous processes, including chronic immune response, the disparity in neuronal circuit dynamics, excitotoxicity, improper myelin function, and reduced neural stem cell (NSC) plasticity and neurogenesis. Even though these aspects are being addressed recently, especially immune and neuronal aspects of AD, there is still a need to address NSC-related issues in less conventional contexts. Perhaps, it is important to determine whether neural stem cells can ever be proliferative and neurogenic when there is amyloid toxicity in the brain: can we elicit neural regeneration in AD conditions? During this thesis, I aimed to answer this question by analyzing the effects of NSC plasticity and neurogenesis on AD pathology using two different animal models of AD. Since, NSCs hold considerable promise in medicine as 'regenerative therapy', where tissue loss could be reversed or the integrity of existing tissue could be improved by employing endogenous NSCs, it is worth hypothesizing that enhancing the neurogenic outcome in AD or healthy brain conditions could enhance resilience and counteract neurodegeneration. To address this aim, I transferred the neurogenic regeneration potential from the adult zebrafish brain to the AD mouse brain. Zebrafish is a well-known model which allowed exploring of the mechanisms of pathology-induced neuro-regeneration in vertebrate brains because of its outstanding neural regeneration potential. Over the years studies focusing on the neuroregenerative ability of adult zebrafish brain after amyloid-mediated neurodegeneration has revealed different molecular mechanisms. One of them is NGFR (nerve growth factor receptor) signaling, which can be utilized to improve NSC plasticity and regeneration. Since NGFR is not expressed in mouse NSCs/astrocytes, I investigated the effect of lentivirus-mediated ectopic overexpression of NGFR in the mouse dentate gyrus (DG) of the hippocampus. I found that the proliferative and neurogenic potentials of the APP/PS1 mouse model of AD were enhanced by the activation of NGFR signaling in mouse astroglia. I further confirmed via single-cell transcriptomics, spatial proteomics, and functional knockdown experiments, that NGFR signaling suppressed reactive astrocyte marker Lipocalin-2 (Lcn2). Furthermore, the blockage of the Lcn2 receptor, solute carrier family 22 member 17 (Slc22a17), recapitulated the neurogenic effects of NGFR. Additionally, long-term NGFR expression in the AD-mouse DG reduced local amyloid-beta plaque load and Tau phosphorylation. Finally, when the postmortem human hippocampi with AD or primary age-related Tauopathy were analyzed for LCN2 expression, I also found it elevated, concomitant to gliosis in astrocytes, indicating the involvement of LCN2 in human AD pathology. This work suggests that NGFR signaling is an evolutionary factor that determines the astrocytes' capacity for neurogenesis. NSCs in zebrafish have naturally activated NGFR signaling throughout their lifespan, which correlates with their lifelong proliferation and neurogenic activity. Transferring this signaling mechanism to mouse models validates the promise of neurogenesis-oriented therapeutic strategies as possible clinical interventions by linking the regulatory function of an autocrine molecular pathway in astroglia to the neurogenic capacity in pathological AD. For my next aim, I further explored the neurogenic potential of zebrafish NSCs to find unknown crucial mechanisms that can be utilized to enhance the NSC plasticity and neurogenesis. During this work, I found that tryptophan's metabolite kynurenic acid (KYNA) inhibited the plasticity of NSCs in the adult zebrafish brain via binding to aryl hydrocarbon receptor 2 (Ahr2). I confirmed that in the case of amyloid toxicity, KYNA synthesis is inhibited by lowering levels of Kynurenine Amino Transferase 2 (KAT2), the primary enzyme that produces KYNA resulting in glial proliferation. Additionally, Ahr2 antagonist increases NSC proliferation, whereas Ahr2 agonists or KYNA decrease it. I further confirm that a subset of Ahr2-expressing zebrafish NSCs do not express other regulatory receptors such as il4r or ngfra, indicating that ahr2-positive NSCs constitute a new subset of neural progenitors that are responsive to amyloid-toxicity. I also found several genes involved in KYNA metabolism or AHR signaling which were differentially expressed in late-onset Alzheimer’s disease (LOAD) by conducting transcriptome-wide association studies (TWAS) in three LOAD brain autopsy cohorts. This finding suggests a strong association between the KYNA/Ahr2 signaling axis and neurogenesis in LOAD. Overall, this work revealed a possible correlation between variations in neurogenesis rate in AD and the antagonistic actions of tryptophan metabolism, potentially via the KYNA/Ahr signaling axis. Collectively, the presented Ph.D. work confirms that enhanced NSC plasticity and neurogenesis could ameliorate AD pathology. Also, it affirms that cellular and molecular pathways learned from zebrafish can be transferred to AD mouse models to investigate whether activating such molecular pathways could initiate neurogenic outcome similar to zebrafish. Overall, these mechanisms could serve as a basis for therapeutic interventions to improve human disease pathology and open up doors for potential drug targets and therapies

Effect of liver x receptor target genes Apolipoprotein E and ATP-binding cassette transporter A1 on beta-amyloid dependent pathology in Alzheimer's disease model mice

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that overtime interferes with daily tasks. Late-onset Alzheimer’s disease (LOAD) is a multifactorial disease with a combination of genetic and environmental risk factors. The APOEε4 allele of Apolipoprotein E (APOE) is the major genetic risk factor for LOAD. However, APOEε3 patients still account for the majority of LOAD cases, suggesting additional genetic, environmental, and lifestyle factors as risk modifiers. We examined the effect of high-fat diet (HFD) and liver x receptor (LXR) agonist T0901317 (T0) in representative mouse models of AD phenotype. LXRs regulate cholesterol and lipoprotein metabolism. ATP-binding cassette transporter A1 (ABCA1) and APOE are major LXR target genes involved in lipid and cholesterol generation and transport and are implicated in AD pathology. We determined that Abca1ko mice have cognitive deficits. Lack of ABCA1 impaired neurite morphology in the CA1 region of the hippocampus. We then examined the effect of HFD on memory deficits and microglia morphology in AD model mice expressing either mouse Apoe or human APOE isoforms. HFD exacerbated cognitive deficits in APP23 mice. Microglia morphology resembled activation state in HFD fed female APOE4 mice, suggesting differential response to diet. Lastly, we examined the effects of T0 on the phenotype and transcriptome of APP/E3 and APP/E4 Abca1 haplo-deficient mice, revealing the ability of T0 to ameliorate APOE4-driven pathological phenotype. These findings suggest that disturbances in cholesterol metabolism may negatively impact AD-related patholoy, HFD exacerbates AD-related pathology, and that T0 treatment ameliorates APOE4-induced AD pathogenesis. These results could have clinical implications on lifestyle or dietary and pharmacological interventions for AD patients. The public health significance of this research supports efforts in developing primary prevention techniques, with the end goal of inhibiting or delaying disease onset, AD-related pathology, and promoting healthy brain aging. Targeting the LXR-ABCA1-APOE regulatory axis could be an effective therapy for individuals at risk of dementia and to treat AD patients regardless of APOE genotype. Further developing studies that better assess cholesterol metabolism genes in AD pathology are essential for modifying guidelines and therapies for those at risk of dementia

Restoring Wnt/β-catenin signaling is a promising therapeutic strategy for Alzheimer's disease.

Alzheimer’s disease (AD) is an aging-related neurological disorder characterized by synaptic loss and dementia. Wnt/β-catenin signaling is an essential signal transduction pathway that regulates numerous cellular processes including cell survival. In brain, Wnt/β-catenin signaling is not only crucial for neuronal survival and neurogenesis, but it plays important roles in regulating synaptic plasticity and blood-brain barrier integrity and function. Moreover, activation of Wnt/β-catenin signaling inhibits amyloid-β production and tau protein hyperphosphorylation in the brain. Critically, Wnt/β-catenin signaling is greatly suppressed in AD brain via multiple pathogenic mechanisms. As such, restoring Wnt/β-catenin signaling represents a unique opportunity for the rational design of novel AD therapies

Studying Glutamatergic Gliotransmission as a Functional Model to Assess Physio-Pathological Conditions and Receptor Cross-Talk

SUMMARY This thesis is divided into two parts; in the first part, we characterized the effect of acute stress on the astrocytic glutamate release in the prefrontal cortex of rat brain. In the second part, we evaluate the interaction of the Oxytocin receptor (OXTR) with both Adenosine receptor A2A and Dopamine D2 receptors in the brain striatum of male rats. First Part: Stress is known to disturb the physiological homeostasis of the body (McEwen et al., 2000). The number of studies demonstrated that stress leads to damage the prefrontal cortex the brain and results in the modulation in the secretion of various neurotransmitters (Karats Oreos and McEwen, 2011, Sousa and Almeida, 2012). Stress was recognized as a predominant risk factor for many diseases, together with cardiovascular, metabolic, and neuropsychiatric diseases. Among the latter, stress interacts with the variable genetic background of vulnerability in the pathogenesis of mood anxiety disorder (Laura et al., 2010). It may be acute or chronic, can involve neuropsychiatric components such as stress, depression, mood, and anxiety (Laura et al., 207) and produces many behavioral and neurochemical changes, as determined in human (De Kloet et al., 2005, Kim &amp; Diamond 2002). Studies in the literature truly indicated that acute stress have an effect on glutamatergic neurotransmission in the prefrontal cortex, inducing changes in glutamate release, receptor and glutamate clearance and metabolism (Popoli et al., 2012: Licznerski and Duman 3013). The effects of acute footshock stress on glutamate release and transmission were still unknown. In this study, we investigated the release of glutamate from astrocytes. The analyses have been performed in the prefrontal cortex (PFC) at different time intervals straight away after 40 min of stress and 6 and 24 hours after stress start, to monitor the early and delayed effects of acute stress on glutamate release. After the acute stress, animals were subjected to sucrose test to distinguish vulnerable and resilient rats. Second Part: There has been a growing interest in the investigation of the role of astrocytes in neurodegenerative and neuropsychiatric diseases and their complex neuron-astrocytes network function. The receptor\u2013receptor interactions (RRI) can have an important function in the signalling transduction pathways. A previous well established study evaluate the interactions between the G-protein coupled receptors of adenosine A2A receptors (A2A) and dopamine D2 receptors (D2) in several experimental models (Ferr\ue9 et al., 2008), while it\u2019s barely investigated in astrocytes. Growing evidence shows that adult striatal astrocytes largely express both D2 and A2A receptors (Cervetto et at., 2017). Moreover, the presence of A2A-D2 heteroreceptor complexes has led to a new perspective of molecular mechanisms involved in Parkinson\u2019s disease (PD), providing novel drug targets. Therefore in the present study, we investigate the physical and functional interactions of A2A and D2 with another G protein-coupled receptors i.e. OXTR in astrocytes processes from adult rat striatum. We also evaluate the effect of this interaction on the astrocytic glutamate release in rat striatum

Investigating the role of hippocampal neurogenesis in Alzheimer's disease.

Neurogenesis is a form of plasticity that occurs constitutively in the neurogenic niches of the mammalian brain. Immature granule cells arising from adult hippocampal neurogenic niche, contribute to unique brain functions in rodents and recent evidences suggests that this process persists throughout life in mammals, including humans. Importantly, studies have also found that hippocampal neurogenesis is impaired in neurodegenerative diseases; most of them concentrate on Alzheimer’s disease. Given the fact the aetiology of this pathology is still unknown and several developed therapies are ineffective for the majority of patients, the study of hippocampal neurogenesis in this condition can provide insights and solutions. In this study we evaluate the neurogenic ability of OXS-N1. This small molecule was discovered as a pro-neurogenic compound through an in vitro phenotypic screening employing primary cell lines from the brain of postnatal mice. It was proven to increase both proliferation and neuronal differentiation of stem and progenitor cells of the two post-natal neurogenic niches. OXS-N1 was subsequently analysed in vivo in wild type mice and in a familial Alzheimer’s disease mouse model (5xFAD). After an evaluation of its effects on memory functions, here we perform an histological analysis on the brain tissues of these mice to assess the impact of OXS-N1 in the subgranular zone. The results we obtained indicate that OXS-N1 does not have the ability to increase the birth or survival of new neurones and neuroblasts, or to induce neural stem cells exit from quiescence. Nevertheless, these data provided an insight in the limitations of OXS-N1 applications in this experimental setting and pointed out the weaknesses of the workflow for the in vivo assessment of the drug’s effects on adult hippocampal neurogenesis