Heterozygous and Homozygous Variants in SORL1 Gene in Alzheimer's Disease Patients: Clinical, Neuroimaging and Neuropathological Findings.

In the last few years, the SORL1 gene has been strongly implicated in the development of Alzheimer's disease (AD). We performed whole-exome sequencing on 37 patients with early-onset dementia or family history suggestive of autosomal dominant dementia. Data analysis was based on a custom panel that included 46 genes related to AD and dementia. SORL1 variants were present in a high proportion of patients with candidate variants (15%, 3/20). We expand the clinical manifestations associated with the SORL1 gene by reporting detailed clinical and neuroimaging findings of six unrelated patients with AD and SORL1 mutations. We also present for the first time a patient with the homozygous truncating variant c.364C>T (p.R122*) in SORL1, who also had severe cerebral amyloid angiopathy. Furthermore, we report neuropathological findings and immunochemistry assays from one patient with the splicing variant c.4519+5G>A in the SORL1 gene, in which AD was confirmed by neuropathological examination. Our results highlight the heterogeneity of clinical presentation and familial dementia background of SORL1-associated AD and suggest that SORL1 might be contributing to AD development as a risk factor gene rather than as a major autosomal dominant gene.

Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur.

Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed aerobic glycolysis. Why engage in inefficient ATP production by glycolysis when energy demand is highest and oxygen is plentiful? Aerobic glycolysis is associated to classic biochemical effects known by the names of Pasteur, Warburg and Crabtree. Here we discuss these three interdependent phenomena in brain cells, in light of high-resolution data of neuronal and astrocytic metabolism in culture, tissue slices and in vivo, acquired with genetically-encoded fluorescent sensors. These sensors are synthetic proteins that can be targeted to specific cell types and subcellular compartments, which change their fluorescence in response to variations in metabolite concentration. A major site of acute aerobic glycolysis is the astrocyte. In this cell, a Crabtree effect triggered by K+ coincides with a Warburg effect mediated by NO, superimposed on a slower longer-lasting Warburg effect caused by glutamate and possibly by NH4+. The compounded outcome is that more fuel (lactate) and more oxygen are made available to neurons, on demand. Meanwhile neurons consume both glucose and lactate, maintaining a strict balance between glycolysis and respiration, commanded by the Na+ pump. We conclude that activity-dependent Warburg and Crabtree effects in brain tissue, and the resulting aerobic glycolysis, do not reflect inefficient energy generation but the marshalling of astrocytes for the purpose of neuronal ATP generation. It remains to be seen whether neurons contribute to aerobic glycolysis under physiological conditions.

Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments.

Monocarboxylate transporter 4 (MCT4) is an H+-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mm, the reported Km of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective Km values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a Km for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous Km estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in high-lactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate.

Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Förster Resonance Energy Transfer Indicator.

The ratio between the cytosolic concentrations of lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a Förster resonance energy transfer (FRET)-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable for use as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in the fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules acetate and ß-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK 293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed robust stability over time and was found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.

Fluid Brain Glycolysis: Limits, Speed, Location, Moonlighting, and the Fates of Glycogen and Lactate.

Glycolysis is the core of intermediate metabolism, an ancient pathway discovered in the heydays of classic biochemistry. A hundred years later, it remains a matter of active research, clinical interest and is not devoid of controversy. This review examines topical aspects of glycolysis in the brain, a tissue characterized by an extreme dependence on glucose. The limits of glycolysis are reviewed in terms of flux control by glucose transporters, intercellular lactate shuttling and activity-dependent glycolysis in astrocytes and neurons. What is the site of glycogen mobilization and aerobic glycolysis in brain tissue? We scrutinize the pervasive notions that glycolysis is fast and that catalysis is channeled through supramolecular assemblies. In brain tissue, most glycolytic enzymes are catalytically silent. What then is their function?

Nanomolar nitric oxide concentrations quickly and reversibly modulate astrocytic energy metabolism.

Nitric oxide (NO) is an intercellular messenger involved in multiple bodily functions. Prolonged NO exposure irreversibly inhibits respiration by covalent modification of mitochondrial cytochrome oxidase, a phenomenon of pathological relevance. However, the speed and potency of NO's metabolic effects at physiological concentrations are incompletely characterized. To this end, we set out to investigate the metabolic effects of NO in cultured astrocytes from mice by taking advantage of the high spatiotemporal resolution afforded by genetically encoded Förster resonance energy transfer (FRET) nanosensors. NO exposure resulted in immediate and reversible intracellular glucose depletion and lactate accumulation. Consistent with cytochrome oxidase involvement, the glycolytic effect was enhanced at a low oxygen level and became irreversible at a high NO concentration or after prolonged exposure. Measurements of both glycolytic rate and mitochondrial pyruvate consumption revealed significant effects even at nanomolar NO concentrations. We conclude that NO can modulate astrocytic energy metabolism in the short term, reversibly, and at concentrations known to be released by endothelial cells under physiological conditions. These findings suggest that NO modulates the size of the astrocytic lactate reservoir involved in neuronal fueling and signaling.

Near-critical GLUT1 and Neurodegeneration.

Recent articles have drawn renewed attention to the housekeeping glucose transporter GLUT1 and its possible involvement in neurodegenerative diseases. Here we provide an updated analysis of brain glucose transport and the cellular mechanisms involved in its acute modulation during synaptic activity. We discuss how the architecture of the blood-brain barrier and the low concentration of glucose within neurons combine to make endothelial/glial GLUT1 the master controller of neuronal glucose utilization, while the regulatory role of the neuronal glucose transporter GLUT3 emerges as secondary. The near-critical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activity-dependent control by neurons may contribute to neurodegeneration. © 2017 Wiley Periodicals, Inc.

Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Inter-Rater Agreement in the Clinical Diagnosis of Cognitive Status: Data from the Neurological Disorders in Central Spain 2 Pilot Study.

BACKGROUND: To assess the diagnostic agreement of cognitive status (dementia, mild cognitive impairment (MCI), normal cognition) among neurologists in the field of neurological disorders in Central Spain 2 study. METHODS: Full medical histories of 30 individuals were provided to 27 neurologists: 9 seniors, 10 juniors and 8 residents. For each case, we were asked to assign a diagnosis of dementia, MCI or normal cognition using the National Institute on Aging-Alzheimer's Association workgroup (NIA-AA) core clinical criteria for all-cause dementia, Winblad et al. criteria for MCI, and analyze intensity and etiology if dementia was diagnosed. Inter-rater agreement was assessed both with percent concordance and non-weighted κ statistics. RESULTS: Overall inter-rater agreement on cognitive status was κ = 0.76 (95% CI 0.65-0.86), being slightly higher among junior neurologists (κ = 0.85, 95% CI 0.73-0.95) than among seniors (κ = 0.71, 95% CI 0.59-0.83) and residents (κ = 0.69, 95% CI 0.54-0.81) but without statistical significance among groups. Dementia severity showed an overall κ of 0.34, 0.44 and 0.64 for mild, moderate and severe dementia respectively. CONCLUSIONS: Substantial agreement was demonstrated for the diagnosis of cognitive status (dementia, MCI and normal cognition) among neurologists of different levels of experience in a population-based epidemiological study using NIA-AA and Winblad et al. CRITERIA: The agreement rate was lower in the diagnosis of dementia severity.

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Lactate-carried Mitochondrial Energy Overflow.

We addressed the question of mitochondrial lactate metabolism using genetically-encoded sensors. The organelle was found to contain a dynamic lactate pool that leads to dose- and time-dependent protein lactylation. In neurons, mitochondrial lactate reported blood lactate levels with high fidelity. The exchange of lactate across the inner mitochondrial membrane was found to be mediated by a high affinity H+-coupled transport system involving the mitochondrial pyruvate carrier MPC. Assessment of electron transport chain activity and determination of lactate flux showed that mitochondria are tonic lactate producers, a phenomenon driven by energization and stimulated by hypoxia. We conclude that an overflow mechanism caps the redox level of mitochondria, while saving energy in the form of lactate.

Fast and reversible stimulation of astrocytic glycolysis by K+ and a delayed and persistent effect of glutamate.

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na(+) overloads elicited by engagement of the Na(+)-glutamate cotransporter with d-aspartate or application of the Na(+) ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K(+) stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K(+) ended immediately but the stimulation by glutamate persisted unabated for >20 min. Both stimulations required an active Na(+)/K(+) ATPase pump. By showing that small rises in extracellular K(+) mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 µM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

Expanding the clinical and genetic spectrum of SQSTM1-related disorders in family with personality disorder and frontotemporal dementia.

Objective:SQSTM1-variants associated with frontotemporal lobar degeneration have been described recently. In this study, we investigated a heterozygous in-frame duplication c.436_462dup p. (Pro146_Cys154dup) in the SQSTM1 gene in a family with a new phenotype characterized by a personality disorder and behavioral variant frontotemporal dementia (bvFTD). We review the literature on frontotemporal dementia (FTD) associated with SQSTM1. Methods: The index case and relatives were described, and a genetic study through Whole Exome Sequencing was performed. The literature was reviewed using Medline and Web of Science. Case reports, case series, and cohort studies were included if they provided information on SQSTM1 mutations associated with FTD. Results: Our patient is a 70-year-old man with a personality disorder since youth, familial history of dementia, and personality disorders with a 10-year history of cognitive decline and behavioral disturbances. A diagnosis of probable bvFTD was established, and the in-frame duplication c.436_462dup in the SQSTM1 gene was identified. Segregation analysis in the family confirmed that both affected sons with personality disorder were heterozygous carriers, but not his healthy 65-year-old brother. A total of 14 publications about 57 patients with SQSTM1-related FTD were reviewed, in which the bvFTD subtype was the main phenotype described (66.6%), with a predominance in men (63%) and positive family history in 61.4% of the cases. Conclusions: We describe a heterozygous in-frame duplication c.436_462dup p.(Pro146_Cys154dup) in the SQSTM1 gene, which affects the zinc-finger domain of p62, in a family with a personality disorder and bvFTD, expanding the genetics and clinical phenotype related to SQSTM1.

Frequency and Characterization of Movement Disorders in Anti-IgLON5 Disease.

OBJECTIVE: Anti-IgLON5 disease is a recently described neurological disease that shares features of autoimmunity and neurodegeneration. Abnormal movements appear to be frequent and important but have not been characterized and are under-reported. Here we describe the frequency and types of movement disorders in a series of consecutive patients with this disease. METHODS: In this retrospective, observational study, the presence and phenomenology of movement disorders were assessed with a standardized clinical questionnaire. Available videos were centrally reviewed by three experts in movement disorders. RESULTS: Seventy two patients were included. In 41 (57%) the main reason for initial consultation was difficulty walking along with one or several concurrent movement disorders. At the time of anti-IgLON5 diagnosis, 63 (87%) patients had at least one movement disorder with a median of three per patient. The most frequent abnormal movements were gait and balance disturbances (52 patients, 72%), chorea (24, 33%), bradykinesia (20, 28%), dystonia (19, 26%), abnormal body postures or rigidity (18, 25%), and tremor (15, 21%). Other hyperkinetic movements (myoclonus, akathisia, myorhythmia, myokymia, or abdominal dyskinesias) occurred in 26 (36%) patients. The craniofacial region was one of the most frequently affected by multiple concurrent movement disorders (23 patients, 32%) including dystonia (13), myorhythmia (6), chorea (4) or myokymia (4). Considering any body region, the most frequent combination of multiple movement disorders consisted of gait instability or ataxia associated with craniofacial dyskinesias or generalized chorea observed in 31(43%) of patients. In addition to abnormal movements, 87% of patients had sleep alterations, 74% bulbar dysfunction, and 53% cognitive impairment. Fifty-five (76%) patients were treated with immunotherapy, resulting in important and sustained improvement of the movement disorders in only seven (13%) cases. CONCLUSIONS: Movement disorders are a frequent and leading cause of initial neurological consultation in patients with anti-IgLON5 disease. Although multiple types of abnormal movements can occur, the most prevalent are disorders of gait, generalized chorea, and dystonia and other dyskinesias that frequently affect craniofacial muscles. Overall, anti-IgLON5 disease should be considered in patients with multiple movement disorders, particularly if they occur in association with sleep alterations, bulbar dysfunction, or cognitive impairment.

A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC.

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

Sleep characteristics in health workers exposed to the COVID-19 pandemic.

INTRODUCTION: The development of sleep disorders, and specifically insomnia, has been linked to the exposure to different stressors. In this line, Coronavirus disease 2019 (COVID-19) outbreak caused by the new coronavirus SARS-CoV-2, has caused a huge impact on our environment, and has exposed healthcare workers to an unprecedented threat. In this study, we try to assess sleep quality and the development of sleep disorders in health personnel directly dedicated to the care of COVID-19 patients at the height of the pandemic, compared to the general population. MATERIALS AND METHODS: A cross-sectional, anonymized, self-reported questionnaire survey was carried out at the "12 de Octubre" Hospital, in Madrid, Spain, during the outbreak of COVID-19, from March 1st to April 30th 2020. We compared two groups, healthcare workers who have treated directly COVID-19 patients versus non-healthcare workers. The questionnaire included demographic data, sleep related aspects, Epworth Sleepiness Scale (ESS), Pittsburgh Sleep Quality Index (PSQI), Insomnia Severity Index (ISI) and 17-items Hamilton Rating Scale (HRS). RESULTS: In total 170 participants completed the questionnaire successfully, 100 healthcare workers and 70 non-healthcare workers. Self-reported insomnia, nightmares, sleepwalking, sleep terrors and PSQI>6 were more frequent in the healthcare group (p < 0,05). Shift work was associated to greater risk when performing multiple logistic regression analysis. CONCLUSIONS: We observed that, during the outbreak of COVID-19, healthcare workers on the front line developed more sleep disturbances than non-healthcare professionals, and they had worse quality of sleep. Special attention should be paid to shift workers. Concrete protection and prevention measures for particularly exposed population should be considered in pandemic situations.

Cognitive Impairment Is a Common Comorbidity in Deceased COVID-19 Patients: A Hospital-Based Retrospective Cohort Study.

We analyzed the frequency of cognitive impairment (CI) in deceased COVID-19 patients at a tertiary hospital in Spain. Among the 477 adult cases who died after admission from March 1 to March 31, 2020, 281 had confirmed COVID-19. CI (21.1% dementia and 8.9% mild cognitive impairment) was a common comorbidity. Subjects with CI were older, tended to live in nursing homes, had shorter time from symptom onset to death, and were rarely admitted to the ICU, receiving palliative care more often. CI is a frequent comorbidity in deceased COVID-19 subjects and is associated with differences in care.

Decreased salivary lactoferrin levels are specific to Alzheimer's disease.

BACKGROUND: Evidences of infectious pathogens in Alzheimer's disease (AD) brains may suggest a deteriorated innate immune system in AD pathophysiology. We previously demonstrated reduced salivary lactoferrin (Lf) levels, one of the major antimicrobial proteins, in AD patients. METHODS: To assess the clinical utility of salivary Lf for AD diagnosis, we examine the relationship between salivary Lf and cerebral amyloid-ß (Aß) load using amyloid-Positron-Emission Tomography (PET) neuroimaging, in two different cross-sectional cohorts including patients with different neurodegenerative disorders. FINDINGS: The diagnostic performance of salivary Lf in the cohort 1 had an area under the curve [AUC] of 0•95 (0•911-0•992) for the differentiation of the prodromal AD/AD group positive for amyloid-PET (PET+) versus healthy group, and 0•97 (0•924-1) versus the frontotemporal dementia (FTD) group. In the cohort 2, salivary Lf had also an excellent diagnostic performance in the health control group versus prodromal AD comparison: AUC 0•93 (0•876-0•989). Salivary Lf detected prodromal AD and AD dementia distinguishing them from FTD with over 87% sensitivity and 91% specificity. INTERPRETATION: Salivary Lf seems to have a very good diagnostic performance to detect AD. Our findings support the possible utility of salivary Lf as a new non-invasive and cost-effective AD biomarker. FUNDING: Instituto de Salud Carlos III (FIS15/00780, FIS18/00118), FEDER, Comunidad de Madrid (S2017/BMD-3700; NEUROMETAB-CM), and CIBERNED (PI2016/01) to E.C.; Spanish Ministry of Economy and Competitiveness (SAF2017-85310-R) to J.L.C., and (PSI2017-85311-P) to M.A.; International Centre on ageing CENIE-POCTEP (0348_CIE_6_E) to M.A.; Instituto de Salud Carlos III (PIE16/00021, PI17/01799), to H.B.