RESUMEN
Accumulated evidence points to the lipocalin apolipoprotein D (ApoD), one of the few genes consistently upregulated upon brain ageing and neurodegeneration, as an endogenous controller of the redox state of cellular and extracellular lipid structures. This biochemical function has downstream consequences as apparently varied as control of glycocalyx and myelin compaction, cell viability upon oxidative stress or modulation of signalling pathways. In spite of this knowledge, it is still unclear if ApoD function requires canonical receptor-mediated transductions systems. This work aims to examine ApoD-cell membrane interaction and its dependence on a proposed ApoD receptor, Basigin. Whole and fractionated membrane preparations from the brain, primary astrocytes, glial and neuronal cell lines, reveal ApoD as a very specific component of particular subtypes of detergent-resistant microdomains (DRMs). ApoD interacts in vitro with neuronal membranes and is stably associated with astrocytic membranes. ApoD associates with DRMs with specific buoyancy properties that co-fractionate with plasma or late-endosome-lysosome markers. A mass spectrometry analysis reveals that these Triton X-114 DRMs contain both plasma membrane and endosomal-lysosomal compartment lipid raft proteins. ApoD-DRM association is maintained under metabolic and acute oxidative stress conditions. However, ApoD-membrane interaction, its internalization and its lipid-antioxidant function do not require the presence of Basigin. This work supports a stable association of ApoD with membranes, independent of Basigin, and provides the basis to fully understand ApoD antioxidant neuroprotective mechanism as a mechanism taking place in specific membrane subdomains.
Asunto(s)
Basigina , Detergentes , Antioxidantes , Apolipoproteínas D/metabolismo , Lipocalinas , Microdominios de Membrana/metabolismoRESUMEN
The mechanisms regulating synapse numbers during development and ageing are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, we demonstrate roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, we find that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and ageing (synapse maintenance) alike. Therefore, our findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer's disease.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Cinesinas/genética , Proteínas de Microfilamentos/genética , Sinapsis/genética , Proteínas tau/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Transporte Axonal , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Movimiento Celular , Demencia/genética , Demencia/metabolismo , Demencia/patología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cinesinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neurogénesis/genética , Neuronas/metabolismo , Neuronas/ultraestructura , Transporte de Proteínas , Transducción de Señal , Sinapsis/metabolismo , Sinapsis/ultraestructura , Proteínas tau/metabolismoRESUMEN
A detailed knowledge of the mechanisms underlying brain aging is fundamental to understand its functional decline and the baseline upon which brain pathologies superimpose. Endogenous protective mechanisms must contribute to the adaptability and plasticity still present in the healthy aged brain. Apolipoprotein D (ApoD) is one of the few genes with a consistent and evolutionarily conserved up-regulation in the aged brain. ApoD protecting roles upon stress or injury are well known, but a study of the effects of ApoD expression in the normal aging process is still missing. Using an ApoD-knockout mouse we analyze the effects of ApoD on factors contributing to the functional maintenance of the aged brain. We focused our cellular and molecular analyses in the cortex and hippocampus at an age representing the onset of senescence where mortality risks are below 25%, avoiding bias towards long-lived animals. Lack of ApoD causes a prematurely aged brain without altering lifespan. Age-dependent hyperkinesia and memory deficits are accompanied by differential molecular effects in the cortex and hippocampus. Transcriptome analyses reveal distinct effects of ApoD loss on the molecular age-dependent patterns of the cortex and hippocampus, with different cell-type contributions to age-regulated gene expression. Markers of glial reactivity, proteostasis, and oxidative and inflammatory damage reveal early signs of aging and enhanced brain deterioration in the ApoD-knockout brain. The lack of ApoD results in an age-enhanced significant reduction in neuronal calcium-dependent functionality markers and signs of early reduction of neuronal numbers in the cortex, thus impinging upon parameters clearly differentiating neurodegenerative conditions from healthy brain aging. Our data support the hypothesis that the physiological increased brain expression of ApoD represents a homeostatic anti-aging mechanism.
Asunto(s)
Envejecimiento/metabolismo , Apolipoproteínas D/fisiología , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Envejecimiento/genética , Envejecimiento/patología , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Envejecimiento Prematuro/patología , Animales , Apolipoproteínas D/deficiencia , Apolipoproteínas D/genética , Conducta Animal , Corteza Cerebral/patología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Femenino , Regulación de la Expresión Génica/fisiología , Hipocampo/patología , Masculino , Ratones Noqueados , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , TranscriptomaRESUMEN
BACKGROUND: A diverse set of neurodegenerative disorders are caused by abnormal extensions of polyglutamine (poly-Q) stretches in various, functionally unrelated proteins. A common feature of these diseases is altered proteostasis. Autophagy induction is part of the endogenous response to poly-Q protein expression. However, if autophagy is not resolved properly, clearance of toxic proteins or aggregates cannot occur effectively. Likewise, excessive autophagy induction can cause autophagic stress and neurodegeneration. The Lipocalins ApoD, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz) are neuroprotectors upon oxidative stress or aging. In this work we test whether these Lipocalins also protect against poly-Q-triggered deterioration of protein quality control systems. RESULTS: Using a Drosophila retinal degeneration model of Type-1 Spinocerebellar Ataxia (SCA1) combined with genetic manipulation of NLaz and GLaz expression, we demonstrate that both Lipocalins protect against SCA1 neurodegeneration. They are part of the endogenous transcriptional response to SCA1, and their effect is non-additive, suggesting participation in a similar mechanism. GLaz beneficial effects persist throughout aging, and appears when expressed by degenerating neurons or by retinal support and glial cells. GLaz gain-of-function reduces cell death and the extent of ubiquitinated proteins accumulation, and decreases the expression of Atg8a/LC3, p62 mRNA and protein levels, and GstS1 induction. Over-expression of GLaz is able to reduce p62 and ubiquitinated proteins levels when rapamycin-dependent and SCA1-dependent inductions of autophagy are combined. In the absence of neurodegeneration, GLaz loss-of-function increases Atg8a/LC3 mRNA and p62 protein levels without altering p62 mRNA levels. Knocking-down autophagy, by interfering with Atg8a or p62 expression or by expressing dominant-negative Atg1/ULK1 or Atg4a transgenes, rescues SCA1-dependent neurodegeneration in a similar extent to the protective effect of GLaz. Further GLaz-dependent improvement is concealed. CONCLUSIONS: This work shows for the first time that a Lipocalin rescues neurons from pathogenic SCA1 degeneration by optimizing clearance of aggregation-prone proteins. GLaz modulates key autophagy genes and lipid-peroxide clearance responsive genes. Down-regulation of selective autophagy causes similar and non-additive rescuing effects. These data suggest that SCA1 neurodegeneration concurs with autophagic stress, and places Lazarillo-related Lipocalins as valuable players in the endogenous protection against the two major contributors to aging and neurodegeneration: ROS-dependent damage and proteostasis deterioration.