Insulin-degrading enzyme (IDE) as a modulator of microglial phenotypes in the context of Alzheimer’s disease and brain aging

The insulin-degrading enzyme (IDE) is an evolutionarily conserved zinc-dependent metallopeptidase highly expressed in the brain, where its specific functions remain poorly understood. Besides insulin, IDE is able to cleave many substrates in vitro, including amyloid beta peptides, making this enzyme a candidate pathophysiological link between Alzheimer's disease (AD) and type 2 diabetes (T2D). These antecedents led us to address the impact of IDE absence in hippocampus and olfactory bulb. A specific induction of microgliosis was found in the hippocampus of IDE knockout (IDE-KO) mice, without any effects in neither hippocampal volume nor astrogliosis. Performance on hippocampal-dependent memory tests is influenced by IDE gene dose in 12-month-old mice. Furthermore, a comprehensive characterization of the impact of IDE haploinsufficiency and total deletion in metabolic, behavioral, and molecular parameters in the olfactory bulb, a site of high insulin receptor levels, reveals an unambiguous barcode for IDE-KO mice at that age. Using wildtype and IDE-KO primary microglial cultures, we performed a functional analysis at the cellular level. IDE absence alters microglial responses to environmental signals, resulting in impaired modulation of phenotypic states, with only transitory effects on amyloid-β management. Collectively, our results reveal previously unknown physiological functions for IDE in microglia that, due to cell-compartment topological reasons, cannot be explained by its enzymatic activity, but instead modulate their multidimensional response to various damaging conditions relevant to aging and AD conditions."Margarita Salas postdoctoral grant for the training of young doctors"/Ministerio de Universidades Predoctoral fellowship/Universidad de Valladolid PID2019-110496RB-C21/Ministerio de Ciencia e Innovación PID2019-110911RB-I00/AEI/Ministerio de Ciencia e Innovación VA086G18/Consejería de Educación, Junta de Castilla y Leó

Dual role of Apolipoprotein D as long-term instructive factor and acute signal conditioning microglial secretory and phagocytic responses

Microglial cells are recognized as very dynamic brain cells, screening the environment and sensitive to signals from all other cell types in health and disease. Apolipoprotein D (ApoD), a lipid-binding protein of the Lipocalin family, is required for nervous system optimal function and proper development and maintenance of key neural structures. ApoD has a cell and state-dependent expression in the healthy nervous system, and increases its expression upon aging, damage or neurodegeneration. An extensive overlap exists between processes where ApoD is involved and those where microglia have an active role. However, no study has analyzed the role of ApoD in microglial responses. In this work, we test the hypothesis that ApoD, as an extracellular signal, participates in the intercellular crosstalk sensed by microglia and impacts their responses upon physiological aging or damaging conditions. We find that a significant proportion of ApoD-dependent aging transcriptome are microglia-specific genes, and show that lack of ApoD in vivo dysregulates microglial density in mouse hippocampus in an age-dependent manner. Murine BV2 and primary microglia do not express ApoD, but it can be internalized and targeted to lysosomes, where unlike other cell types it is transiently present. Cytokine secretion profiles and myelin phagocytosis reveal that ApoD has both long-term pre-conditioning effects on microglia as well as acute effects on these microglial immune functions, without significant modification of cell survival. ApoD-triggered cytokine signatures are stimuli (paraquat vs. Aβ oligomers) and sex-dependent. Acute exposure to ApoD induces microglia to switch from their resting state to a secretory and less phagocytic phenotype, while long-term absence of ApoD leads to attenuated cytokine induction and increased myelin uptake, supporting a role for ApoD as priming or immune training factor. This knowledge should help to advance our understanding of the complex responses of microglia during aging and neurodegeneration, where signals received along our lifespan are combined with damage-triggered acute signals, conditioning both beneficial roles and limitations of microglial functions

Lipid-binding proteins in brain health and disease

A proper lipid management is paramount for a healthy brain. Lipid homeostasis alterations are known to be causative or risk factors for many neurodegenerative diseases, or key elements in the recovery from nervous system injuries of different etiology. In addition to lipid biogenesis and catabolism, non-enzymatic lipid-binding proteins play an important role in brain function and maintenance through aging. Among these types of lipoproteins, apolipoprotein E has received much attention due to the relationship of particular alleles of its gene with the risk and progression of Alzheimer's disease. However, other lipid-binding proteins whose role in lipid homeostasis and control are less known need to be brought to the attention of both researchers and clinicians. The aim of this review is to cover the knowledge of lipid-managing proteins in the brain, with particular attention to new candidates to be relevant for brain function and health.This work was supported by grants to MG and DS from Ministerio de Ciencia e Innovación, MICINN (Grant No. BFU2015-68149-R), and Consejería de Educación Junta Castilla y León (Grant No. VA086G18), co-financed by European Regional Development Fund. MC-G was supported by a University of Valladolid Ph.D. fellowship (call#2016).Peer reviewe

The MTT-formazan assay: Complementary technical approaches and in vivo validation in Drosophila larvae

The MTT assay was the first widely accepted method to assess cytotoxicity and cell viability. However, there is controversy on whether this indicator is a useful tool. In this work we intend to expand the interpretability of the MTT study by its combination with widely used cellular biology techniques. We propose complementary approaches to the colorimetric assay, based on the use of measurements in three different settings: confocal microscopy, multi-well plate assay and flow cytometry. Using confocal microscopy, we confirmed that MTT uptake and reduction by cells is a time-dependent process, and that formazan accumulates in round-shaped organelles. Quantitative measurements with a multi-well fluorimeter combined with nuclear staining result in a useful method, yielding a ratio between formazan production and cell number that informs about the average cell metabolic state. We also found that flow cytometry is a suitable technique to measure MTT reduction in large cell populations. When assaying the effect of an oxidizing agent such as paraquat (PQ), this approach allows for the distinction of subpopulations of cells with different reducing power. Finally, we prove that it is feasible to monitor MTT reduction in an in vivo model, the Drosophila larvae, without affecting its survival rate. Formazan accumulates exclusively in the larval fat body, confirming its lipid solubility. The methods explored in this work expand the MTT potential as a useful tool to provide information of the physiological state of cells and organisms.This work was supported by grants to MDG and DS (Ministerio de Ciencia e Innovación (MICINN) grant BFU2015-68149-R, co-financed by European Regional Development Fund). MCG was supported by a University of Valladolid fellowship to young researchers (call#2016). RPM was supported by a Junta de Castilla y León (JCyL) fellowship to young researchers (call#EDU/1883/2013), financed by the European Social Fund, Operational Programme for Castilla y León and managed by Consejería de Educación (JCyL).Peer reviewe

Apolipoprotein D-mediated preservation of lysosomal function promotes cell survival and delays motor impairment in Niemann-Pick type A disease

ts, contributing to NPA lysosomes vulnerability. Exogenously added ApoD reduces NPA-prompted lysosomal permeabilization and alkalinization, reverts lipid peroxides accumulation, and significantly increases NPA cell survival. ApoD administered simultaneously to sphingomyelin overload results in complete rescue of cell survival. Our results reveal that ApoD protection of lysosomal integrity counteracts NPA pathology. ApoD supplementation could significantly delay not only the progression of NPA disease, but also of other LSDs through its beneficial effects in lysosomal functional maintenance.This work was supported by grants to M.D.G. and D.S. (Ministerio de Ciencia e Innovación (MICINN) grants BFU2011-23978 and BFU2015-68149-R) and to M.D.L. (Ministerio de Ciencia e Innovación SAF2017-87698-R), as well as a grant from the National Institutes of Health to E.H.S. (5-37-HD28607). R.P-M. was supported by a Junta de Castilla y León (JCyL) fellowship to young researchers (call#EDU/1883/2013), financed by the European Social Fund, Operational Programme and managed by Consejería de Educación (JCyL). M.C-G. was supported by a University of Valladolid PhD fellowship (call#2016).Peer reviewe

Insulin-degrading enzyme (IDE) as a modulator of microglial phenotypes in the context of Alzheimer’s disease and brain aging

Abstract The insulin-degrading enzyme (IDE) is an evolutionarily conserved zinc-dependent metallopeptidase highly expressed in the brain, where its specific functions remain poorly understood. Besides insulin, IDE is able to cleave many substrates in vitro, including amyloid beta peptides, making this enzyme a candidate pathophysiological link between Alzheimer's disease (AD) and type 2 diabetes (T2D). These antecedents led us to address the impact of IDE absence in hippocampus and olfactory bulb. A specific induction of microgliosis was found in the hippocampus of IDE knockout (IDE-KO) mice, without any effects in neither hippocampal volume nor astrogliosis. Performance on hippocampal-dependent memory tests is influenced by IDE gene dose in 12-month-old mice. Furthermore, a comprehensive characterization of the impact of IDE haploinsufficiency and total deletion in metabolic, behavioral, and molecular parameters in the olfactory bulb, a site of high insulin receptor levels, reveals an unambiguous barcode for IDE-KO mice at that age. Using wildtype and IDE-KO primary microglial cultures, we performed a functional analysis at the cellular level. IDE absence alters microglial responses to environmental signals, resulting in impaired modulation of phenotypic states, with only transitory effects on amyloid-β management. Collectively, our results reveal previously unknown physiological functions for IDE in microglia that, due to cell-compartment topological reasons, cannot be explained by its enzymatic activity, but instead modulate their multidimensional response to various damaging conditions relevant to aging and AD conditions