Maria D. Ganfornina

Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress

Many nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila, Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up‐regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids.

Molecular identification of Kvα subunits that contribute to the oxygen-sensitive K+ current of chemoreceptor cells of the rabbit carotid body

Rabbit carotid body (CB) chemoreceptor cells possess a fast‐inactivating K+ current that is specifically inhibited by hypoxia. We have studied the expression of Kvα subunits, which might be responsible for this current. RT‐PCR experiments identified the expression of Kv1.4, Kv3.4, Kv4.1 and Kv4.3 mRNAs in the rabbit CB. There was no expression of Kv3.3 or Kv4.2 transcripts. Immunocytochemistry with antibodies to tyrosine hydroxylase (anti‐TH) and to specific Kv subunits revealed the expression of Kv3.4 and Kv4.3 in chemoreceptor cells, while Kv1.4 was only found in nerve fibres. Kv4.1 mRNA was also found in chemoreceptor cells following in situ hybridization combined with anti‐TH antibody labelling. Kv4.1 and Kv4.3 appeared to be present in all chemoreceptor cells, but Kv3.4 was only expressed in a population of them. Electrophysiological experiments applying specific toxins or antibodies demonstrated that both Kv3.4 and Kv4.3 participate in the oxygen‐sensitive K+ current of chemoreceptor cells. However, toxin application experiments confirmed a larger contribution of members of the Kv4 subfamily. [Ca2+]i measurements under hypoxic conditions and immunocytochemistry experiments in dispersed CB cells demonstrated the expression of Kv3.4 and Kv4.3 in oxygen‐sensitive cells; the presence of Kv3.4 in the chemoreceptor cell membrane was not required for the response to low PO2. In summary, three Kv subunits (Kv3.4, Kv4.1 and Kv4.3) may be involved in the fast‐inactivating outward K+ current of rabbit CB chemoreceptor cells. The homogeneous distribution of the Kv4 subunits in chemoreceptor cells, along with their electrophysiological properties, suggest that Kv4.1, Kv4.3, or their heteromultimers, are the molecular correlate of the oxygen‐sensitive K+ channel.

Control of Metabolic Homeostasis by Stress Signaling Is Mediated by the Lipocalin NLaz

Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways—such as Jun-N-terminal Kinase (JNK) signaling—repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. Here, we identify a new regulatory mechanism by which JNK influences metabolism systemically. We show that JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan—phenotypes that are consistent with reduced IIS activity. Accordingly, we find that NLaz represses IIS activity in larvae and adult flies. Our results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis.

Altered lipid metabolism in a Drosophila model of Friedreich's ataxia

Abstract Friedreichs ataxia (FRDA) is the most common form of autosomal recessive ataxia caused by a deficit in the mitochondrial protein frataxin. Although demyelination is a common symptom in FRDA patients, no multicellular model has yet been developed to study the involvement of glial cells in FRDA. Using the recently established RNAi lines for targeted suppression of frataxin in Drosophila, we were able to study the effects of general versus glial-specific frataxin downregulation. In particular, we wanted to study the interplay between lowered frataxin content, lipid accumulation and peroxidation and the consequences of these effects on the sensitivity to oxidative stress and fly fitness. Interestingly, ubiquitous frataxin reduction leads to an increase in fatty acids catalyzing an enhancement of lipid peroxidation levels, elevating the intracellular toxic potential. Specific loss of frataxin in glial cells triggers a similar phenotype which can be visualized by accumulating lipid droplets in glial cells. This phenotype is associated with a reduced lifespan, an increased sensitivity to oxidative insult, neurodegenerative effects and a serious impairment of locomotor activity. These symptoms fit very well with our observation of an increase in intracellular toxicity by lipid peroxides. Interestingly, co-expression of a Drosophila apolipoprotein D ortholog (glial lazarillo) has a strong protective effect in our frataxin models, mainly by controlling the level of lipid peroxidation. Our results clearly support a strong involvement of glial cells and lipid peroxidation in the generation of FRDA-like symptoms.

ApoD, a glia-derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury.

Glial cells are a key element to the process of axonal regeneration, either promoting or inhibiting axonal growth. The study of glial derived factors induced by injury is important to understand the processes that allow or preclude regeneration, and can explain why the PNS has a remarkable ability to regenerate, while the CNS does not. In this work we focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glial cells in the PNS and CNS. ApoD expression is strongly induced upon PNS injury, but its role has not been elucidated. Here we show that ApoD is required for: (1) the maintenance of peripheral nerve function and tissue homeostasis with age, and (2) an adequate and timely response to injury. We study crushed sciatic nerves at two ages using ApoD knock‐out and transgenic mice over‐expressing human ApoD. The lack of ApoD decreases motor nerve conduction velocity and the thickness of myelin sheath in intact nerves. Following injury, we analyze the functional recovery, the cellular processes, and the protein and mRNA expression profiles of a group of injury‐induced genes. ApoD helps to recover locomotor function after injury, promoting myelin clearance, and regulating the extent of angiogenesis and the number of macrophages recruited to the injury site. Axon regeneration and remyelination are delayed without ApoD and stimulated by excess ApoD. The mRNA and protein expression profiles reveal that ApoD is functionally connected in an age‐dependent manner to specific molecular programs triggered by injury.

Apolipoprotein D mediates autocrine protection of astrocytes and controls their reactivity level, contributing to the functional maintenance of paraquat-challenged dopaminergic systems

The study of glial derived factors induced by injury and degeneration is important to understand the nervous system response to deteriorating conditions. We focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glia and strongly induced upon aging, injury or neurodegeneration. Here we study ApoD function in the brain of wild type and ApoD‐KO mice by combining in vivo experiments with astrocyte cultures. Locomotor performance, dopamine concentration, and gene expression levels in the substantia nigra were assayed in mice treated with paraquat (PQ). The regulation of ApoD transcription, a molecular screening of oxidative stress (OS)‐related genes, cell viability and oxidation status, and the effects of adding human ApoD were tested in astrocyte cultures. We demonstrate that (1) ApoD is required for an adequate locomotor performance, modifies the gene expression profile of PQ‐challenged nigrostriatal system, and contributes to its functional maintenance; (2) ApoD expression in astrocytes is controlled by the OS‐responsive JNK pathway; (3) ApoD contributes to an autocrine protecting mechanism in astrocytes, avoiding peroxidated lipids accumulation and altering the PQ transcriptional response of genes involved in ROS managing and the inflammatory response to OS; (4) Addition of human ApoD to ApoD‐KO astrocytes promotes survival through a mechanism accompanied by protein internalization and modulation of astroglial reactivity. Our data support that ApoD contributes to the endurance of astrocytes and decreases their reactivity level in vitro and in vivo. ApoD function as a maintenance factor for astrocytes would suffice to explain the observed protection by ApoD of OS‐vulnerable dopaminergic circuits in vivo.

Comparative gene expression profile of mouse carotid body and adrenal medulla under physiological hypoxia

The carotid body (CB) is an arterial chemoreceptor, bearing specialized type I cells that respond to hypoxia by closing specific K+ channels and releasing neurotransmitters to activate sensory axons. Despite having detailed information on the electrical and neurochemical changes triggered by hypoxia in CB, the knowledge of the molecular components involved in the signalling cascade of the hypoxic response is fragmentary. This study analyses the mouse CB transcriptional changes in response to low PO2 by hybridization to oligonucleotide microarrays. The transcripts were obtained from whole CBs after mice were exposed to either normoxia (21% O2), or physiological hypoxia (10% O2) for 24 h. The CB transcriptional profiles obtained under these environmental conditions were subtracted from the profile of control non‐chemoreceptor adrenal medulla extracted from the same animals. Given the common developmental origin of these two organs, they share many properties but differ specifically in their response to O2. Our analysis revealed 751 probe sets regulated specifically in CB under hypoxia (388 up‐regulated and 363 down‐regulated). These results were corroborated by assessing the transcriptional changes of selected genes under physiological hypoxia with quantitative RT‐PCR. Our microarray experiments revealed a number of CB‐expressed genes (e.g. TH, ferritin and triosephosphate isomerase) that were known to change their expression under hypoxia. However, we also found novel genes that consistently changed their expression under physiological hypoxia. Among them, a group of ion channels show specific regulation in CB: the potassium channels Kir6.1 and Kcnn4 are up‐regulated, while the modulatory subunit Kcnab1 is down‐regulated by low PO2 levels.

Expression pattern of the lipocalin Apolipoprotein D during mouse embryogenesis

Apolipoprotein D (ApoD) is a secreted protein that belongs to the lipocalin family. We describe the expression pattern of ApoD during mouse embryogenesis by in situ hybridization using RNA probes. ApoD is expressed at E9 in mesenchymal cells in the rombencephalic-mesencephalic region. At E9.5 the cephalic ApoD-positive cells appear in the mesenchyme, and at later stages (starting at E10.5) ApoD expression is seen in meninges. Within the neuroepithelium, ApoD is expressed in pericytes surrounding brain and spinal cord capillaries from E10.5 to birth. Other places of expression of ApoD are the mesenchyme surrounding the olfactory epithelium and semicircular canals, as well as chondroblasts of skull and vertebrae.

Molecular characterization and developmental expression pattern of the chicken apolipoprotein D gene: implications for the evolution of vertebrate lipocalins.

The insect Lazarillo and the mammalian apolipoprotein D (ApoD) are orthologous members of the lipocalin protein family. We report the cloning and embryonic expression of chicken ApoD, the first molecularly characterized nonmammalian ApoD. We also report the ApoD expression in mouse during postnatal development and some novel aspects of the expression of the paralogous lipocalin prostaglandin D‐synthase (PGDS) and discuss these results in view of the lipocalin family evolution in vertebrates. ApoD is expressed in subsets of central nervous system (CNS) neurons and glia during late chicken embryogenesis. Contrary to mouse ApoD, no expression appears in neural crest‐derived cephalic mesenchyme and blood vessel pericytes. Also, ApoD is expressed in developing chicken feathers. These expressions are corroborated by quantitative reverse transcriptase‐polymerase chain reaction profiles. ApoD is expressed during mouse postnatal development in a subset of CNS neurons, astrocytes and oligodendrocytes, but also in meninges and pericytes. Chicken PGDS is expressed in brain meninges and perivascular cells. Our results suggest that the amniote last common ancestor expressed ApoD and PGDS in the brain during embryogenesis. ApoD appears restricted to ectodermal derivatives, whereas PGDS is expressed by derivatives of the three germ layers. Developmental Dynamics 232:191–199, 2005.

Apolipoprotein D alters the early transcriptional response to oxidative stress in the adult cerebellum

J. Neurochem. (2011) 117, 949–960.