Complicaciones centrales en modelos de enfermedad de alzheimer y diabetes: papel de la empagliflozina

La enfermedad de Alzheimer (EA) es la causa más común de demencia. Además de las características neuropatológicas clásicas, otras complicaciones, como el proceso neuroinflamatorio, también han cobrado relevancia en los últimos años. Sin embargo, la valoración in vivo de la inflamación y la microglía resulta especialmente difícil. La microscopía multifotón (MMF) permite, con alta resolución, la detección in vivo de células de microglía individuales en el cerebro de modelos complejos de forma crónica. En este trabajo hemos realizado una revisión de estudios que emplean la MMF en ratones modelo de EA. Mediante MMF es posible evaluar el papel dual de la microglía en la EA: favoreciendo el aclaramiento de amiloide en estadios iniciales y contribuyendo a su depósito cuando el proceso se hace crónico. Otros trabajos de MMF han revelado que la inmunoterapia con anticuerpos anti β-amiloide (βA), como enfoque terapéutico clave para el tratamiento de la EA, contribuye a la eliminación de βA mediante la activación de microglía. En conjunto los estudios basados en MMF pueden ayudar a esclarecer el papel de la microglía en el proceso inflamatorio y los cambios que se producen a nivel central a medida que progresa la EA. Además del proceso neuroinflamatorio, el concepto de neurodegeneración también se ha ampliado para incluir otras alteraciones menos clásicas asociadas a la EA, como las alteraciones en la proliferación y neurogenesis. Dada la relación entre la EA y las enfermedades metabólicas, y en concreto la diabetes mellitus tipo 2 (DM2), hemos analizado los procesos de proliferación celular y neurogénesis mediante doble inmunotinción con 5´-bromo-2´-desoxiuridina (BrdU) y doblecortina (DCX) en tres modelos diferentes de EA y alteraciones metabólicas. Hemos utilizado un modelo de EA tratado con streptozotozina (STZ) para inducir diabetes mellitus tipo 1 (DM1) (ratón APP/PS1-STZ), un modelo de EA y prediabetes inducido por el tratamiento a largo plazo con una dieta rica en grasas (DRG) (ratón APP/PS1-DRG) y un modelo mixto de EA-DM2 (ratón APP/PS1xdb/db). Observamos una reducción general en la proliferación celular y neurogénesis tras la administración de STZ en la zona subventricular (ZSV), corteza e hipocampo. Mientras, en los ratones APP/PS1-DRG observamos un incremento de este proceso en la corteza. Finalmente, detectamos un aumento de la proliferación celular y la neurogénesis en la ZSV y giro dentado de los ratones APP/PS1xdb/db a las 14 y 26 semanas de edad, acompañado de un ligero aumento en la proliferación en la corteza. Aunque estudios recientes han mostrado la expresión de DCX en células T-CD8+ y molécula ionizada adaptadora de unión a calcio 1 (Iba1+) en la proximidad de las placas seniles (PS), en nuestro estudio apenas observamos células DCX+/CD8+ y no detectamos ninguna célula DCX+/Iba1+. También observamos que los parámetros metabólicos, como el peso corporal y los niveles de glucosa, son buenos predictores de las alteraciones en los niveles de βA cortical y del hipocampo. Es más, los parámetros metabólicos también se correlacionan con la proliferación celular y neurogénesis en los ratones APP/PS1xdb/db. Nuestros resultados apoyan que las alteraciones metabólicas, características de la DM2, pueden predecir las complicaciones cerebrales propias de la EA a la par que se produce una exacerbación de la patología cuando ambas enfermedades se establecen concomitantemente. A la complejidad y a su relación con la DM2, debe sumarse el hecho de que la EA a día de hoy no tiene tratamiento exitoso. Esto hace necesaria la búsqueda de alternativas terapéuticas. La relación EA-DM2 apoya el estudio de fármacos antidiabéticos en la patología tipo Alzheimer. La empagliflozina (EMP) es un inhibidor del transportador sodio-glucosa 2 (iSGLT2) recientemente comercializado, capaz de controlar la patología metabólica y de reducir las muertes de causa cardiovascular asociadas a la DM2. Teniendo en cuenta estas consideraciones, hemos tratado el modelo mixto de EA-DM2 (ratón APP/PS1xdb/db) con EMP durante 22 semanas. Nuestros resultados muestran como la EMP ayuda a mantener los niveles de insulina en los ratones diabéticos. A nivel central, limita el adelgazamiento cortical y la pérdida neuronal, reduciendo la atrofia cerebral. La carga de hemorragias y de microglía también disminuye tras el tratamiento con EMP, mejorando la patología de pequeño vaso y limitando la inflamación. De igual modo, la carga de PS y la fosforilación de tau se reducen, mejorando la patología Alzheimer. Todo esto se acompaña de una mejora de los déficits cognitivos que presentan los ratones APP/PS1xdb/db. En conjunto, nuestros resultados apoyan el posible papel de la EMP en la patología asociada a la EA. La situación crítica de los pacientes de Alzheimer hace necesario su abordaje desde diferentes ángulos. En los últimos años han cobrado relevancia características de la enfermedad, como alteraciones del proceso inflamatorio central, y los estudios con MMF pueden ayudar a esclarecer el papel fisiológico y patológico del proceso neuroinflamatorio y la microglía en el cerebro de EA. Otras alteraciones, como los procesos de proliferación y neurogénesis, también se ven afectados en la EA y en modelos mixtos de EA-DM. La exacerbación de la patología central en nuestro modelo mixto (APP/PS1xdb/db) subraya la estrecha relación entre ambas enfermedades y un posible efecto sinérgico. Teniendo en cuenta este agravamiento generalizado cuando ambas patologías coexisten, y la estrecha relación EA-DM, es posible que el tratamiento con fármacos antidiabéticos pueda limitar los daños a nivel central. Así la administración de EMP mejora la patología tipo Alzheimer, inflamatoria y vascular en los animales APP/PS1xdb/db, abriendo la puerta a continuar estos estudios

Empagliflozin reduces brain pathology in Alzheimer’s disease and type 2 diabetes

Alzheimer’s disease (AD) and type 2 diabetes (T2D): More than 55 million people suffer from dementia, and it is expected that over 150 million people will suffer from this disease by 2050. AD is the most common type of dementia and while aging remains the main risk factor to suffer it, previous studies have also shown that metabolic disorders, and T2D specifically, are also major contributors (Wang et al., 2012). The prevalence of diabetes has reached 537 million people worldwide and these figures are expected to keep rising (Ahmad et al., 2022). All things considered, both diseases are a great challenge for health

Amyloid beta and diabetic pathology cooperatively stimulate cytokine expression in an Alzheimer's mouse model

Background Diabetes is a risk factor for developing Alzheimer's disease (AD); however, the mechanism by which diabetes can promote AD pathology remains unknown. Diabetes results in diverse molecular changes in the brain, including dysregulation of glucose metabolism and loss of cerebrovascular homeostasis. Although these changes have been associated with increased A beta pathology and increased expression of glial activation markers in APPswe/PS1dE9 (APP/PS1) mice, there has been limited characterization, to date, of the neuroinflammatory changes associated with diabetic conditions. Methods To more fully elucidate neuroinflammatory changes associated with diabetes that may drive AD pathology, we combined the APP/PS1 mouse model with either high-fat diet (HFD, a model of pre-diabetes), the genetic db/db model of type 2 diabetes, or the streptozotocin (STZ) model of type 1 diabetes. We then used a multiplexed immunoassay to quantify cortical changes in cytokine proteins. Results Our analysis revealed that pathology associated with either db/db, HFD, or STZ models yielded upregulation of a broad profile of cytokines, including chemokines (e.g., MIP-1 alpha, MIP-1 beta, and MCP-1) and pro-inflammatory cytokines, including IL-1 alpha, IFN-gamma, and IL-3. Moreover, multivariate partial least squares regression analysis showed that combined diabetic-APP/PS1 models yielded cooperatively enhanced expression of the cytokine profile associated with each diabetic model alone. Finally, in APP/PS1xdb/db mice, we found that circulating levels of A beta 1-40, A beta 1-42, glucose, and insulin all correlated with cytokine expression in the brain, suggesting a strong relationship between peripheral changes and brain pathology. Conclusions Altogether, our multiplexed analysis of cytokines shows that Alzheimer's and diabetic pathologies cooperate to enhance profiles of cytokines reported to be involved in both diseases. Moreover, since many of the identified cytokines promote neuronal injury, A beta and tau pathology, and breakdown of the blood-brain barrier, our data suggest that neuroinflammation may mediate the effects of diabetes on AD pathogenesis. Therefore, strategies targeting neuroinflammatory signaling, as well as metabolic control, may provide a promising strategy for intervening in the development of diabetes-associated AD

Empagliflozin reduces vascular damage and cognitive impairment in a mixed murine model of Alzheimer's disease and type 2 diabetes

Background Both Alzheimer's disease (AD) and type 2 diabetes (T2D) share common pathological features including inflammation, insulin signaling alterations, or vascular damage. AD has no successful treatment, and the close relationship between both diseases supports the study of antidiabetic drugs to limit or slow down brain pathology in AD. Empagliflozin (EMP) is a sodium-glucose co-transporter 2 inhibitor, the newest class of antidiabetic agents. EMP controls hyperglycemia and reduces cardiovascular comorbidities and deaths associated to T2D. Therefore, we have analyzed the role of EMP at the central level in a complex mouse model of AD-T2D. Methods We have treated AD-T2D mice (APP/PS1xdb/db mice) with EMP 10 mg/kg for 22 weeks. Glucose, insulin, and body weight were monthly assessed. We analyzed learning and memory in the Morris water maze and the new object discrimination test. Postmortem brain assessment was conducted to measure brain atrophy, senile plaques, and amyloid-beta levels. Tau phosphorylation, hemorrhage burden, and microglia were also measured in the brain after EMP treatment. Results EMP treatment helped to maintain insulin levels in diabetic mice. At the central level, EMP limited cortical thinning and reduced neuronal loss in treated mice. Hemorrhage and microglia burdens were also reduced in EMP-treated mice. Senile plaque burden was lower, and these effects were accompanied by an amelioration of cognitive deficits in APP/PS1xdb/db mice. Conclusions Altogether, our data support a feasible role for EMP to reduce brain complications associated to AD and T2D, including classical pathological features and vascular disease, and supporting further assessment of EMP at the central level

Alzheimer's Disease and Diabetes: Role of Diet, Microbiota and Inflammation in Preclinical Models

Alzheimer's disease (AD) is the most common cause of dementia. Epidemiological studies show the association between AD and type 2 diabetes (T2DM), although the mechanisms are not fully understood. Dietary habits and lifestyle, that are risk factors in both diseases, strongly modulate gut microbiota composition. Also, the brain-gut axis plays a relevant role in AD, diabetes and inflammation, through products of bacterial metabolism, like short-chain fatty acids. We provide a comprehensive review of current literature on the relation between dysbiosis, altered inflammatory cytokines profile and microglia in preclinical models of AD, T2DM and models that reproduce both diseases as commonly observed in the clinic. Increased proinflammatory cytokines, such as IL-1 beta and TNF-alpha, are widely detected. Microbiome analysis shows alterations in Actinobacteria, Bacteroidetes or Firmicutes phyla, among others. Altered alpha- and beta-diversity is observed in mice depending on genotype, gender and age; therefore, alterations in bacteria taxa highly depend on the models and approaches. We also review the use of pre- and probiotic supplements, that by favoring a healthy microbiome ameliorate AD and T2DM pathologies. Whereas extensive studies have been carried out, further research would be necessary to fully understand the relation between diet, microbiome and inflammation in AD and T2DM

Antidiabetic polypill improves central pathology and cognitive impairment in a mixed model of Alzheimer's disease and type 2 diabetes

Type 2 diabetes (T2D) is an important risk factor to suffer dementia, being Alzheimer's disease (AD) as the most common form. Both AD and T2D are closely related to aging and with a growing elderly population it might be of relevance to explore new therapeutic approaches that may slow or prevent central complications associated with metabolic disorders. Therefore, we propose the use of the antidiabetic polypill (PP), a pharmacological cocktail, commonly used by T2D patients that include metformin, aspirin, simvastatin, and an angiotensin-converting enzyme inhibitor. In order to test the effects of PP at the central level, we have long-term treated a new mixed model of AD-T2D, the APP/PS1xdb/db mouse. We have analyzed AD pathological features and the underlying specific characteristics that relate AD and T2D. As expected, metabolic alterations were ameliorated after PP treatment in diabetic mice, supporting a role for PP in maintaining pancreatic activity. At central level, PP reduced T2D-associated brain atrophy, showing both neuronal and synaptic preservation. Tau and amyloid pathologies were also reduced after PP treatment. Furthermore, we observed a reduction of spontaneous central bleeding and inflammation after PP treatment in diabetic mice. As consequence, learning and memory processes were improved after PP treatment in AD, T2D, and AD-T2D mice. Our data provide the basis to further analyze the role of PP, as an alternative or adjuvant, to slow down or delay the central complications associated with T2D and AD

Effects of classical PKC activation on hippocampal neurogenesis and cognitive performance: mechanism of action

Hippocampal neurogenesis has widely been linked to memory and learning performance. New neurons generated from neural stem cells (NSC) within the dentate gyrus of the hippocampus (DG) integrate in hippocampal circuitry participating in memory tasks. Several neurological and neuropsychiatric disorders show cognitive impairment together with a reduction in DG neurogenesis. Growth factors secreted within the DG promote neurogenesis. Protein kinases of the protein kinase C (PKC) family facilitate the release of several of these growth factors, highlighting the role of PKC isozymes as key target molecules for the development of drugs that induce hippocampal neurogenesis. PKC activating diterpenes have been shown to facilitate NSC proliferation in neurogenic niches when injected intracerebroventricularly. We show in here that long-term administration of diterpene ER272 promotes neurogenesis in the subventricular zone and in the DG of mice, affecting neuroblasts differentiation and neuronal maturation. A concomitant improvement in learning and spatial memory tasks performance can be observed. Insights into the mechanism of action reveal that this compound facilitates classical PKCα activation and promotes transforming growth factor alpha (TGFα) and, to a lesser extent, neuregulin release. Our results highlight the role of this molecule in the development of pharmacological drugs to treat neurological and neuropsychiatric disorders associated with memory loss and a deficient neurogenesis.Ministerio de Ciencia, Innovación y Universidades de España (MICINN) RTI-2018-099908-B-C21 y RTI-2018-099908-B-C22Ministerio de Ciencia, Innovación y Universidades de España y Fondos FEDER de la Unión Europea (MICINN/FEDER) BFU2016-75038RConsejería de Economía, Conocimiento, Empresas y Universidades de la Junta de Andalucía y Fondos FEDER. FEDER-UCA18-10664

Accelerated amyloid angiopathy and related vascular alterations in a mixed murine model of Alzheimer´s disease and type two diabetes

Amyloid; Multiphoton microscopy; PrediabetesAmiloide; Microscòpia multifotònica; PrediabetisAmiloide; Microscopía multifotónica; PrediabetesBackground While aging is the main risk factor for Alzheimer´s disease (AD), emerging evidence suggests that metabolic alterations such as type 2 diabetes (T2D) are also major contributors. Indeed, several studies have described a close relationship between AD and T2D with clinical evidence showing that both diseases coexist. A hallmark pathological event in AD is amyloid-β (Aβ) deposition in the brain as either amyloid plaques or around leptomeningeal and cortical arterioles, thus constituting cerebral amyloid angiopathy (CAA). CAA is observed in 85–95% of autopsy cases with AD and it contributes to AD pathology by limiting perivascular drainage of Aβ. Methods To further explore these alterations when AD and T2D coexist, we have used in vivo multiphoton microscopy to analyze over time the Aβ deposition in the form of plaques and CAA in a relevant model of AD (APPswe/PS1dE9) combined with T2D (db/db). We have simultaneously assessed the effects of high-fat diet-induced prediabetes in AD mice. Since both plaques and CAA are implicated in oxidative-stress mediated vascular damage in the brain, as well as in the activation of matrix metalloproteinases (MMP), we have also analyzed oxidative stress by Amplex Red oxidation, MMP activity by DQ™ Gelatin, and vascular functionality. Results We found that prediabetes accelerates amyloid plaque and CAA deposition, suggesting that initial metabolic alterations may directly affect AD pathology. T2D significantly affects vascular pathology and CAA deposition, which is increased in AD-T2D mice, suggesting that T2D favors vascular accumulation of Aβ. Moreover, T2D synergistically contributes to increase CAA mediated oxidative stress and MMP activation, affecting red blood cell velocity. Conclusions Our data support the cross-talk between metabolic disease and Aβ deposition that affects vascular integrity, ultimately contributing to AD pathology and related functional changes in the brain microvasculature.University of Cadiz Predoctoral Fellowship (CHB). This study is part of the current project (RECOGNISED; Clinical Trials gov registration no. NCT04281186) funded by the European Commission (H2020 programme-GA 847749) focusing on common mechanisms in the pathogenesis of diabetic retinopathy, brain pathology and cognitive impairment, with special interest in the neurovascular unit, in the T2D population. Agencia Estatal de Investigacion. Ministerio de Ciencia e Innovacion. Programa Estatal de Generacion de Conocimiento y Fortalecimiento Cientifico y Tecnologico del Sistema de I + D + i y del Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, del Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion (PID2020-115499RB-I00/AEI/10.130 39/501100011033). Programa Estatal de I + D + I orientada a los Retos de la Sociedad (BFU 2016-75038-R), financed by the Agencia Estatal de Investigacion (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER), Ministerio de Economia y Competitividad. Proyectos de I + D + i, en regimen de concurrencia competitiva, destinadas a las universidades y entidades publicas de investigacion calificadas como agentes del Sistema Andaluz del Conocimiento, en el ambito del Plan Andaluz de Investigacion, Desarrollo e Innovación (PAIDI 2020). Andalucia se mueve con Europa (P20-00928)

Liraglutide Reduces Vascular Damage, Neuronal Loss, and Cognitive Impairment in a Mixed Murine Model of Alzheimer's Disease and Type 2 Diabetes

Alzheimer's disease is the most common form of dementia, and epidemiological studies support that type 2 diabetes (T2D) is a major contributor. The relationship between both diseases and the fact that Alzheimer's disease (AD) does not have a successful treatment support the study on antidiabetic drugs limiting or slowing down brain complications in AD. Among these, liraglutide (LRGT), a glucagon-like peptide-1 agonist, is currently being tested in patients with AD in the Evaluating Liraglutide in Alzheimer's Disease (ELAD) clinical trial. However, the effects of LRGT on brain pathology when AD and T2D coexist have not been assessed. We have administered LRGT (500 mu g/kg/day) to a mixed murine model of AD and T2D (APP/PS1xdb/db mice) for 20 weeks. We have evaluated metabolic parameters as well as the effects of LRGT on learning and memory. Postmortem analysis included assessment of brain amyloid-beta and tau pathologies, microglia activation, spontaneous bleeding and neuronal loss, as well as insulin and insulin-like growth factor 1 receptors. LRGT treatment reduced glucose levels in diabetic mice (db/db and APP/PS1xdb/db) after 4 weeks of treatment. LRGT also helped to maintain insulin levels after 8 weeks of treatment. While we did not detect any effects on cortical insulin or insulin-like growth factor 1 receptor m-RNA levels, LRGT significantly reduced brain atrophy in the db/db and APP/PS1xdb/db mice. LRGT treatment also rescued neuron density in the APP/PS1xdb/db mice in the proximity (p = 0.008) far from amyloid plaques (p < 0.001). LRGT reduced amyloid plaque burden in the APP/PS1 animals (p < 0.001), as well as A beta aggregates levels (p = 0.046), and tau hyperphosphorylation (p = 0.009) in the APP/PS1xdb/db mice. Spontaneous bleeding was also ameliorated in the APP/PS1xdb/db animals (p = 0.012), and microglia burden was reduced in the proximity of amyloid plaques in the APP/PS1 and APP/PS1xdb/db mice (p < 0.001), while microglia was reduced in areas far from amyloid plaques in the db/db and APP/PS1xdb/db mice (p < 0.001). This overall improvement helped to rescue cognitive impairment in AD-T2D mice in the new object discrimination test (p < 0.001) and Morris water maze (p < 0.001). Altogether, our data support the role of LRGT in reduction of associated brain complications when T2D and AD occur simultaneously, as regularly observed in the clinical arena.CH-B: predoctoral fellowship. University of Cadiz. PA-M: predoctoral fellowship. Instituto de Investigacion Biomedica de la Provincia de Cadiz (INIBICA). MG-A: Agencia Estatal de Investigacion. Ministerio de Ciencia e Innovacion. Programa Estatal de Generacion de Conocimiento y Fortalecimiento Cientifico y Tecnologico del Sistema de I C D C i y del Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, del Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion 2017-2020 (PID2020115499RB-I0). Programa Estatal de I C D C I orientada a los Retos de la Sociedad (BFU 2016-75038-R), financed by the Agencia Estatal de Investigacion (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER), Ministerio de Ciencia, Innovacion y Universidades. Subvencion para la financiacion de la Investigacion y la Innovacion Biomedica y en Ciencias de la Salud en el Marco de la Iniciativa Territorial Integrada 2014-2020 para la Provincia de Cadiz. Consejeria de Salud. Junta de Andalucia. Union Europea, financed by the Fondo de Desarrollo Regional (FEDER) (PI-0008-2017)

Effects of classical PKC activation on hippocampal neurogenesis and cognitive performance: mechanism of action

Hippocampal neurogenesis has widely been linked to memory and learning performance. New neurons generated from neural stem cells (NSC) within the dentate gyrus of the hippocampus (DG) integrate in hippocampal circuitry participating in memory tasks. Several neurological and neuropsychiatric disorders show cognitive impairment together with a reduction in DG neurogenesis. Growth factors secreted within the DG promote neurogenesis. Protein kinases of the protein kinase C (PKC) family facilitate the release of several of these growth factors, highlighting the role of PKC isozymes as key target molecules for the development of drugs that induce hippocampal neurogenesis. PKC activating diterpenes have been shown to facilitate NSC proliferation in neurogenic niches when injected intracerebroventricularly. We show in here that long-term administration of diterpene ER272 promotes neurogenesis in the subventricular zone and in the DG of mice, affecting neuroblasts differentiation and neuronal maturation. A concomitant improvement in learning and spatial memory tasks performance can be observed. Insights into the mechanism of action reveal that this compound facilitates classical PKC alpha activation and promotes transforming growth factor alpha (TGF alpha) and, to a lesser extent, neuregulin release. Our results highlight the role of this molecule in the development of pharmacological drugs to treat neurological and neuropsychiatric disorders associated with memory loss and a deficient neurogenesis