Dr. Eduardo Castaño
Jefe de Laboratorio
Envejecimiento cerebral y Neurodegeneración Versión en español
Subject
The progressive accumulation of misfolded proteins in the brain (amyloid beta, tau) is one the most relevant abnormal processes in AD. Our hypothesis is that aging affects key mechanisms involved in the prevention and clearance of protein aggregates in certain neuronal populations. We further propose that once initiated, the accretion of amyloid beta and tau aggregates triggers an irreversible self-promoting cascade with a negative and protracted impact upon basic neuronal processes. Among these, we are interested in the characterization of changes in energy metabolism, including the utilization of glucose and mitochondrial function. In a line of applied research, we are interested in defining correlations between diet, nutrition and cognitive impairment at early stages of the pathological process both in an animal model and in patients with early AD. These correlations include the assessment of peripheral biomarkers with possible predictive value for disease progression. In the same direction, we aim to identify which brain metabolic pathways associated with cognitive decline may be modified by interventions upon intestinal microbiota. Finally, we explore the optimization of the predictive value of genetic polymorphisms associated with ancestry effect in the Argentinean population as compared with other studies in Caucasians.
Approach
We use three study models to perform our basic research: Transgenic Drosophila melanogaster expressing Aβ in the secretory pathway of neurons, transgenic rats expressing in CNS neurons a mutant of amyloid precursor protein (Mc Gill R-Thy1 APP K670N, K670N, M671L) and human neuronal cell lines transfected with Aβ. In Drosophila, we study the unfolded protein response in the endoplasmic reticulum (ER-UPR) by negative geotaxis, immunochemistry of specific proteins and gene expression by quantitative PCR with aging and Aβ expression. In addition, the Drosophila model was used to perform a genetic screen of ~200 lines carrying small chromosomal deletions to identify modifiers that enhance or reduce Aβ neurotoxicity and their impact on specific metabolic pathways by nuclear magnetic resonance (NMR). In McGill Tg rats and primary cultured neurons from their brains we study the correlation between Aβ accumulation, memory impairment and basic alterations of mitochondrial function, including the interaction of this organelle with the ER. We performed a combination of oximetry with a Seahorse Xfe24, real time FRET of fluorescent protein constructs as organelle cross-linkers activated by rifampicin, and mitochondrial lipidomics in primary neurons. For the preclinical study of correlations between nutrition, metabolic pathways and cognitive decline we use McGill tg rats in early and late stages of the pathological process. We analyze the effect of a defined diet rich in lipids and sugars upon behavior and correlations with pro-inflammatory cytokines in plasma, brain and fat tissue; markers of oxidative/nitrosative stress in plasma and brain, insulin signaling in brain and changes in microbiota/fecal metabolome by 16S rRNA sequencing and mass spectrometry. These and other variables with a potential as biomarkers will be further studied in AD patients. The studies of genetic polymorphisms, ancestry and predictive value are performed with more than 1000 samples from “probable AD” patients and controls within the AGA-ALZAR_PRONADIAL consortium established in Argentina. DNA-NGS sequencing and bioinformatic-statistical analysis is performed in collaboration with consortia and academic institutions in the European Union.
Advances
We have demonstrated that in Drosophila, protection from Aβ-mediated brain toxicity and a delay in its accumulation depend on IRE1-XP1 signaling and its balance with the PERK branch of the ER-UPR, which show antagonistic effects. While XBP1 is strongly protective, PERK promotes Aβ neurotoxicity. In addition, we have established that aging promotes a progressive decline in the XBP1 response at the level of transcription of effector protein chaperones whose molecular mechanisms remain to be determined. The genetic screen allowed the identification of modifiers of Aβ toxicity, as assessed by negative geotaxis and confirmed with lines expressing specific interference RNAs. The partial deficiency of the gene encoding the enzyme 4-hydroxyl-phenyl-piruvate-dioxygenase (HPD) was associated with a higher accumulation of Aβ and the increment of age-dependent neurotoxicity. HPD is the second enzyme in Tyrosine degradation and its genetic defect causes the human disease called Tyrosinemia type III. Yet, our results of NMR metabolome in a neuronal cell line expressing Aβ suggest that the effect of HPD deficiency may be mediated by dysregulation of the pentose pathway and oxidative stress independent of tyrosine metabolism (unpublished results). The studies on mitochondrial function in McGill tg rats showed that the accumulation of APP/Aβ induced a reduction in reserve respiratory capacity and enzymatic activity of complex I in brain synaptosomes. In addition, by using primary neuronal cultures from this animal model we demonstrated a reduction in close contacts (≤10 nm) between the outer mitochondrial membrane and the ER that may affect inter-organelle transport of lipids that are essential for the optimal function of mitochondria. A diet rich in fat and sugars as compared to standard diet.
Mc Gill tg rats fed with a diet rich in fat and sugars showed a higher impairment of cognitive decline, the accumulation of toxic Aβ species in the hippocampus, a reduction in neuroprotective signaling pathways and increased nitrosative damage.
Regarding our clinical applied project, we have consolidated the largest cohort of patients with “probable AD” in Latinoamerica and developed a predictive model of genetic risk for AD in persons with ancestry admixture that may be more accurate than those currently based in Caucasians.
The progressive accumulation of misfolded proteins in the brain (amyloid beta, tau) is one the most relevant abnormal processes in AD. Our hypothesis is that aging affects key mechanisms involved in the prevention and clearance of protein aggregates in certain neuronal populations. We further propose that once initiated, the accretion of amyloid beta and tau aggregates triggers an irreversible self-promoting cascade with a negative and protracted impact upon basic neuronal processes. Among these, we are interested in the characterization of changes in energy metabolism, including the utilization of glucose and mitochondrial function. In a line of applied research, we are interested in defining correlations between diet, nutrition and cognitive impairment at early stages of the pathological process both in an animal model and in patients with early AD. These correlations include the assessment of peripheral biomarkers with possible predictive value for disease progression. In the same direction, we aim to identify which brain metabolic pathways associated with cognitive decline may be modified by interventions upon intestinal microbiota. Finally, we explore the optimization of the predictive value of genetic polymorphisms associated with ancestry effect in the Argentinean population as compared with other studies in Caucasians.
Approach
We use three study models to perform our basic research: Transgenic Drosophila melanogaster expressing Aβ in the secretory pathway of neurons, transgenic rats expressing in CNS neurons a mutant of amyloid precursor protein (Mc Gill R-Thy1 APP K670N, K670N, M671L) and human neuronal cell lines transfected with Aβ. In Drosophila, we study the unfolded protein response in the endoplasmic reticulum (ER-UPR) by negative geotaxis, immunochemistry of specific proteins and gene expression by quantitative PCR with aging and Aβ expression. In addition, the Drosophila model was used to perform a genetic screen of ~200 lines carrying small chromosomal deletions to identify modifiers that enhance or reduce Aβ neurotoxicity and their impact on specific metabolic pathways by nuclear magnetic resonance (NMR). In McGill Tg rats and primary cultured neurons from their brains we study the correlation between Aβ accumulation, memory impairment and basic alterations of mitochondrial function, including the interaction of this organelle with the ER. We performed a combination of oximetry with a Seahorse Xfe24, real time FRET of fluorescent protein constructs as organelle cross-linkers activated by rifampicin, and mitochondrial lipidomics in primary neurons. For the preclinical study of correlations between nutrition, metabolic pathways and cognitive decline we use McGill tg rats in early and late stages of the pathological process. We analyze the effect of a defined diet rich in lipids and sugars upon behavior and correlations with pro-inflammatory cytokines in plasma, brain and fat tissue; markers of oxidative/nitrosative stress in plasma and brain, insulin signaling in brain and changes in microbiota/fecal metabolome by 16S rRNA sequencing and mass spectrometry. These and other variables with a potential as biomarkers will be further studied in AD patients. The studies of genetic polymorphisms, ancestry and predictive value are performed with more than 1000 samples from “probable AD” patients and controls within the AGA-ALZAR_PRONADIAL consortium established in Argentina. DNA-NGS sequencing and bioinformatic-statistical analysis is performed in collaboration with consortia and academic institutions in the European Union.
Advances
We have demonstrated that in Drosophila, protection from Aβ-mediated brain toxicity and a delay in its accumulation depend on IRE1-XP1 signaling and its balance with the PERK branch of the ER-UPR, which show antagonistic effects. While XBP1 is strongly protective, PERK promotes Aβ neurotoxicity. In addition, we have established that aging promotes a progressive decline in the XBP1 response at the level of transcription of effector protein chaperones whose molecular mechanisms remain to be determined. The genetic screen allowed the identification of modifiers of Aβ toxicity, as assessed by negative geotaxis and confirmed with lines expressing specific interference RNAs. The partial deficiency of the gene encoding the enzyme 4-hydroxyl-phenyl-piruvate-dioxygenase (HPD) was associated with a higher accumulation of Aβ and the increment of age-dependent neurotoxicity. HPD is the second enzyme in Tyrosine degradation and its genetic defect causes the human disease called Tyrosinemia type III. Yet, our results of NMR metabolome in a neuronal cell line expressing Aβ suggest that the effect of HPD deficiency may be mediated by dysregulation of the pentose pathway and oxidative stress independent of tyrosine metabolism (unpublished results). The studies on mitochondrial function in McGill tg rats showed that the accumulation of APP/Aβ induced a reduction in reserve respiratory capacity and enzymatic activity of complex I in brain synaptosomes. In addition, by using primary neuronal cultures from this animal model we demonstrated a reduction in close contacts (≤10 nm) between the outer mitochondrial membrane and the ER that may affect inter-organelle transport of lipids that are essential for the optimal function of mitochondria. A diet rich in fat and sugars as compared to standard diet.
Mc Gill tg rats fed with a diet rich in fat and sugars showed a higher impairment of cognitive decline, the accumulation of toxic Aβ species in the hippocampus, a reduction in neuroprotective signaling pathways and increased nitrosative damage.
Regarding our clinical applied project, we have consolidated the largest cohort of patients with “probable AD” in Latinoamerica and developed a predictive model of genetic risk for AD in persons with ancestry admixture that may be more accurate than those currently based in Caucasians.