Jose Antonio Rodriguez-Navarro

Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance

Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.

Trehalose ameliorates dopaminergic and tau pathology in parkin deleted/tau overexpressing mice through autophagy activation.

Tauopathies are neurodegenerative diseases, sporadic or familial, mainly characterized by dementia and parkinsonism associated to atrophy of the frontotemporal cortex and the basal ganglia, with deposition of abnormal tau in brain. Hereditary tauopathies are related with mutations of the tau gene. Up to the present, these diseases have not been helped by any disease-modifying treatment, and patients die a few years after the onset of symptoms. We have developed and characterized a mouse model of tauopathy with parkinsonism, overexpressing human mutated tau protein with deletion of parkin (PK(-/-)/Tau(VLW)). At 3 months of age, these mice present abnormal dopamine-related behavior, severe dropout of dopamine neurons in the ventral midbrain, reduced dopamine levels in the striatum and abundant phosphorylated tau-positive neuritic plaques, neurofibrillary tangles, astrogliosis, and, at 12 months old, plaques of murine beta-amyloid in the hippocampus. Trehalose is a natural disaccharide that increases the removal of abnormal proteins through enhancement of autophagy. In this work, we tested if 1% trehalose in the drinking water reverts the PK(-/-)/Tau(VLW) phenotype. The treatment with trehalose of 3-month-old PK(-/-)/Tau(VLW) mice for 2.5 months reverted the dropout of dopamine neurons, which takes place in the ventral midbrain of vehicle treated PK(-/-)/Tau(VLW) and the reduced dopamine-related proteins levels in the midbrain and striatum. The number of phosphorylated tau-positive neuritic plaques and the levels of phosphorylated tau decreased, as well as astrogliosis in brain regions. The autophagy markers in the brain, the autophagic vacuoles isolated from the liver, and the electron microscopy data indicate that these effects of trehalose are mediated by autophagy. The treatment with trehalose for 4 months of 3-month-old PK(-/-)/Tau(VLW) mice maintained the amelioration of the tau pathology and astrogliosis but failed to revert DA-related pathology in the striatum. Furthermore, the 3-week treatment with trehalose of 14-month-old PK(-/-)/Tau(VLW) mice, at the limit of their life expectancy, improved the motor behavior and anxiety of these animals, and reduced their levels of phosphorylated tau and the number of murine beta-amyloid plaques. Trehalose is neuroprotective in this model of tauopathy. Since trehalose is free of toxic effects at high concentrations, this study opens the way for clinical studies of the effects of trehalose in human tauopathies.

Inhibitory effect of dietary lipids on chaperone-mediated autophagy

Cytosolic proteins can be selectively delivered to lysosomes for degradation through a type of autophagy known as chaperone-mediated autophagy (CMA). CMA contributes to intracellular quality control and to the cellular response to stress. Compromised CMA has been described in aging and in different age-related disorders. CMA substrates cross the lysosomal membrane through a translocation complex; consequently, changes in the properties of the lysosomal membrane should have a marked impact on CMA activity. In this work, we have analyzed the impact that dietary intake of lipids has on CMA activity. We have found that chronic exposure to a high-fat diet or acute exposure to a cholesterol-enriched diet both have an inhibitory effect on CMA. Lysosomes from livers of lipid-challenged mice had a marked decrease in the levels of the CMA receptor, the lysosome-associated membrane protein type 2A, because of loss of its stability at the lysosomal membrane. This accelerated degradation of lysosome-associated membrane protein type 2A, also described as the mechanism that determines the decline in CMA activity with age, results from its increased mobilization to specific lipid regions at the lysosomal membrane. Comparative lipidomic analyses revealed qualitative and quantitative changes in the lipid composition of the lysosomal membrane of the lipid-challenged animals that resemble those observed with age. Our findings identify a previously unknown negative impact of high dietary lipid intake on CMA and underscore the importance of diet composition on CMA malfunction in aging.

Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimers disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimers disease.

Autophagy and lipids: tightening the knot

The degradation of intracellular components in lysosomes, also known as autophagy, participates in a broad range of cellular functions from cellular quality control to cellular remodeling or as mechanism of defense against cellular aggressors. In this review, we focus on the role of autophagy as an alternative source of cellular energy, particularly important when nutrients are scarce. Almost since the discovery of autophagy, it has been known that amino acids obtained through the breakdown of proteins in lysosomes are essential to maintaining the cellular energetic balance during starvation. However, it is only recently that the ability of autophagy to mobilize intracellular lipid stores as an additional source of energy has been described. Autophagy contributes thus to modulating the amount of cellular lipids and allows cells to adapt to lipogenic stimuli. Interestingly, this interplay between autophagy and lipid metabolism is bidirectional, as changes in the intracellular lipid content also contribute to modulating autophagic activity. In this review, we describe the recent findings on the contribution of autophagy to lipid metabolism in different tissues and the consequences that impairments in autophagy have on cellular physiology. In addition, we comment on the regulatory role that lipid molecules and their modifying enzymes play on different steps of the autophagic process.

The effects of parkin suppression on the behaviour, amyloid processing, and cell survival in APP mutant transgenic mice.

Parkin suppression induces accumulation of beta-amyloid in mutant tau mice. We studied the effect of parkin suppression on behaviour and brain pathology in APP(swe) mutant mice. We produced double mutant mice with human mutated APP(swe)+partial (hemizygote) or total (homozygote) deletion of Park-2 gene. We studied the development, behaviour, brain histology, and biochemistry of 12- and 16-month-old animals in 6 groups of mice, with identical genetic background: wild-type (WT), APP(swe) overexpressing (APP), hemizygote and homozygote deletion of Park-2 (PK(+/-) and PK(-/-), respectively), and double mutants (APP/PK(+/-) and APP/PK(-/-)). APP mice have reduced weight gain, decreased motor activity, and reduced number of entrances and of arm alternation in the Y-maze, abnormalities which were partially or completely normalized in APP/PK(+/-) and APP/PK(-/-) mice. The double mutants had similar number of mutant human APP transgene copies than the APP and levels of 40 and 80 kDa proteins; but both of them, APP/PK(+/-) and APP/PK(-/-) mice, had less plaques in cortex and hippocampus than the APP mice. APP mutant mice had increased apoptosis, proapoptotic Bax/Bcl2 ratios, and gliosis, but these death-promoting factors were normalized in APP/PK(+/-) and APP/PK(-/-) mice. APP mutant mice had an increased number of tau immunoreactive neuritic plaques in the cerebral cortex as well as increased levels of total and phosphorylated tau protein, and these changes were partially normalized in APP/PK(+/-) heterozygotic and homozygotic APP/PK(-/-) mice. Compensatory protein-degrading systems such as HSP70, CHIP, and macroautophagy were increased in APP/PK(+/-) and APP/PK(-/-). Furthermore, the chymotrypsin- and trypsin-like proteasome activities, decreased in APP mice in comparison with WT, were normalized in the APP/PK(-/-) mice. We proposed that partial and total suppression of parkin triggers compensatory mechanisms, such as chaperone overexpression and increased autophagy, which improved the behavioural and cellular phenotype of APP(swe) mice.

Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.

Autophagy and amino acid metabolism in the brain: implications for epilepsy

Autophagy is a catabolic pathway responsible for the maintenance of the tissue and organism homeostasis. Several amino acids regulate autophagic activity in different tissues, such as liver and muscle, but much less is known about this regulation in the brain. The lack of autophagy in neurons leads to a strong neurodegenerative phenotype and epileptic disorders. We summarize the current knowledge about the regulation of autophagy mediated by amino acids and how macroautophagy could serve as source of amino acids. We review the contribution of macroautophagy in the brain physiology and pathology emphasizing the relevancy of the proper control of amino acid levels such as glutamate and GABA in the brain due to its role as neurotransmitters and energy source. Furthermore, we discuss how malfunction in autophagy may result in pathological consequences, because many genetic epileptic disorders are related to signaling or metabolic pathways controlling both macroautophagy and amino acid metabolism in the brain.

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates.

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions.

Unique Features of Neuronal Autophagy: Considerations for Therapeutic Targeting

Autophagy is an essential catabolic pathway responsible for the maintenance of organismal homeostasis. Degradation of damaged organelles and proteinaceous aggregates predominantly takes place via autophagy and a proper function of autophagy is vital for cellular surveillance. Given their postmitotic nature, neurons are particularly vulnerable to stress and, consequently, robust housekeeping systems are required to guarantee the adequate functionality and viability of neurons. A vast literature links defective autophagic function to neurodegenerative diseases and dietary/pharmacological activation of autophagy has been proposed as potential strategy to fight these diseases. Here we summarize the recent progress on the research of neuronal autophagy highlighting the unique features of autophagy in neurons. In the last section, we discuss about therapeutic strategies modulating autophagy to preserve neuronal surveillance during aging.