The neuroprotective actions of oestradiol and oestrogen receptors

Recent discoveries have shown that both hormonal and brain-derived oestradiol have neuroprotective effects. This Review provides a comprehensive review of the multiple cell types, receptors and signalling cascades that underlie oestradiol-mediated neuroprotection.

Abstract

Hormones regulate homeostasis by communicating through the bloodstream to the body's organs, including the brain. As homeostatic regulators of brain function, some hormones exert neuroprotective actions. This is the case for the ovarian hormone 17β-oestradiol, which signals through oestrogen receptors (ERs) that are widely distributed in the male and female brain. Recent discoveries have shown that oestradiol is not only a reproductive hormone but also a brain-derived neuroprotective factor in males and females and that ERs coordinate multiple signalling mechanisms that protect the brain from neurodegenerative diseases, affective disorders and cognitive decline.

Key points

• Oestradiol is both a sex steroid hormone and a neurosteroid that is locally synthesized in the brain. Both hormonal oestradiol and brain-derived oestradiol are neuroprotective.
• Oestradiol synthesis in the brain is rapidly regulated in neurons by synaptic activity. In turn, brain-derived oestradiol regulates synaptic plasticity, adult neurogenesis, reproductive behaviour, aggressive behaviour, pain processing, affect and cognition.
• Under pathological conditions, the expression of aromatase, the enzyme that produces oestradiol, is enhanced in neurons and induced de novo in astrocytes as an endogenous neuroprotective mechanism. Inhibition or silencing of brain aromatase increases gliosis and neurodegeneration after brain injury.
• The neuroprotective actions of oestradiol are mediated by two oestrogen receptors (ERs) located in the cell nucleus, ERα and ERβ, and by ERs located in the membrane, including ERα, ERβ, G protein-coupled ER and Gαq protein-coupled ER.
• The ERs coordinate various neuroprotective signalling mechanisms, some of which are complementary and some of which are redundant. These include the regulation of transcription by nuclear ERs and the regulation of the activity of different kinases by membrane ERs.
• Membrane and intracellular ERs also contribute to the interaction of oestradiol signalling with the signalling of other neuroprotective factors, such as brain-derived neurotrophic factor, insulin-like growth factor 1, WNT and Notch.
• Further studies are necessary to determine the role of neuronal and non-neuronal cells in the coordination of oestradiol-mediated neuroprotective signalling mechanisms. The influence of sex and age on these mechanisms should also be studied.
• A promising research direction aims to determine the role of metabolic homeostatic regulation in the protective actions of oestradiol, particularly in chronic neurodegenerative diseases.
• Further research should also explore alternatives to oestradiol therapy, including new ligands for ERs and modulators of brain oestradiol synthesis.

Introduction

17β-oestradiol (oestradiol) is a steroid hormone that is synthesized in a series of consecutive enzymatic steps that begins with the conversion of cholesterol into pregnenolone in the mitochondria (Box 1). The final enzymatic step, the conversion of testosterone into oestradiol, is catalysed by the enzyme aromatase (also known as oestrogen synthase and encoded by the CYP19A1 gene; Box 1). Oestradiol, as a hormone, is mainly produced in the ovary. Its levels in the plasma change during development and puberty, fluctuate during the menstrual cycle (or the oestrous cycle in rodents) and suddenly decline with menopause. Oestradiol is also locally synthesized in different tissues, including bone, adipose and nervous tissues, in both males and females. Indeed, an important concept that has progressively developed in recent decades is the idea that the brain is a steroidogenic organ¹ that expresses the molecules and enzymes necessary for the conversion of cholesterol into steroids such as progesterone, testosterone and oestradiol (Box 1). Therefore, the brain is a target both for steroids derived from peripheral steroidogenic glands and for neurosteroids (steroids synthesized in neural cells)¹. Neurosteroids control various neurobiological processes, including cognition, stress, anxiety, depression, aggressiveness, body temperature, blood pressure, locomotion, feeding behaviour and sexual behaviour². Brain steroidogenesis is regulated independently of peripheral steroidogenesis, and plasma steroid levels do not directly reflect brain steroid levels^{3, 4}. However, after ovariectomy in female rats and orchidectomy in males, the brain adapts the levels of steroids in a sex- and region-specific manner⁵, suggesting a compensatory adaptation of brain steroidogenesis in response to gonadal steroid deprivation.

Box 1: Brain steroidogenesis  

The gonads, the adrenal glands and the placenta are the classical steroidogenic endocrine glands in the body. However, other organs, including the brain, also have steroidogenic activity. The first step in steroidogenesis is the conversion of cholesterol to pregnenolone in the inner mitochondrial membrane (IMM, see the figure, left panel)¹⁴³. Several proteins are involved in this step, including steroidogenic acute regulatory protein (StAR), translocator protein of 18 KDa (TSPO) and cytochrome P450 cholesterol side chain cleavage enzyme (P450scc). StAR and TSPO are involved in the transport of cholesterol from the outer mitochondrial membrane (OMM) to the IMM, which is a highly regulated step. In the IMM, cholesterol is transformed into pregnenolone by P450scc, the first steroidogenic enzyme. StAR, TSPO and P450scc form a multimolecular complex, termed the transduceosome, together with other proteins, including the 31-kDa voltage-dependent anion-selective channel (VDAC) in the OMM and the 35-kDa mitochondrial adenine nucleotide translocator (ANT) in the IMM¹⁴³. Pregnenolone is then converted to other steroids through a series of consecutive steps (see the figure, right panel) in the endoplasmic reticulum.

 

 

The rodent brain expresses StAR, TSPO, P450scc and the steroidogenic enzymes necessary to produce oestradiol from cholesterol². Several of these enzymes have also been identified in the human brain¹⁴³. The steroids produced in the nervous system, known as neurosteroids¹, act through various mechanisms, including the direct modulation of the channel properties of synaptic neurotransmitter receptors, such as the NMDA receptor and the GABA_A receptor, by the progesterone metabolite 3α,5α-tetrahydroprogesterone (also known as allopregnanolone)^{144, 145}. Some neurosteroids, such as oestradiol, regulate neural function, behaviour, cognition and affect, and have neuroprotective actions^{144, 146}. The term neuroactive steroid is also currently used in the literature to refer to steroids with activity in the nervous system, independently of whether they are peripheral hormones, neurosteroids or synthetic steroids¹⁴⁴.

 

HSD, hydroxysteroid dehydrogenase.

 

Oestradiol is both a peripheral hormone and a neurosteroid. Oestradiol is synthesized in the adult male and female brain, where it acts as an autocrine factor or a paracrine factor that, under physiological conditions regulates synaptic plasticity, adult neurogenesis, reproductive behaviour, aggressive behaviour, pain processing, affect and cognition (Box 2). The actions of brain-derived oestradiol contribute to the maintenance of brain homeostasis, and recent findings indicate that it is also a neuroprotective factor, decreasing neural damage in animal models of brain injury and neurodegenerative diseases. In this Review, we describe recent experimental evidence that emphasizes the role of brain oestradiol synthesis as an endogenous neuroprotective mechanism.

Box 2: Role of brain aromatase under physiological conditions

The enzyme aromatase, which converts androgens into oestrogens, is widely expressed in neurons from different brain regions of male and female animals. These include brain areas involved in reproductive control and brain regions related to memory, emotion and cognitive processing. For example, in humans, aromatase immunoreactivity has been detected in, among other brain structures, pyramidal neurons and some interneurons in the cerebral cortex and the hippocampus¹⁴⁷. In addition, aromatase has been detected at synapses in birds and mammals^{148, 149, 150}.

 

In the brains of birds and mammals, aromatase is involved in the regulation of synaptogenesis and synaptic plasticity^{151, 152}, neurogenesis^{153, 154}, reproductive behaviour¹⁵⁵, aggressive behaviour¹⁵⁵, pain processing^{156, 157}, auditory processing¹⁵⁸, social communication¹⁵⁸, affect and cognition^{159, 160, 161}.

 

Recent findings indicate that oestradiol synthesis in the hippocampus is regulated by gonadotropin-releasing hormone¹⁶², which may coordinate oestradiol synthesis in the brain and the ovary. Furthermore, aromatase activity, and therefore oestradiol production, is rapidly regulated in the brain by glutamatergic synapses¹⁵⁵. The synaptic location of aromatase, together with the rapid regulation of its activity by neurotransmitters, suggests that brain-derived oestradiol acts as a rapid neuromodulator in synaptic circuits^{155, 163}. Indeed, rapid changes in brain aromatase activity are linked to swift changes in synaptic function, neuronal activity and behaviour^{155, 158}. Thus, local oestradiol synthesis in the brain, modulated by rapid changes in aromatase activity, seems to play an important part in the regulation of brain function and behaviour under physiological conditions.

In parallel with the advances in our knowledge of the roles of brain-derived oestradiol in neural function and neuroprotection, considerable recent progress has been made in establishing the underlying molecular mechanisms. In particular, it has been discovered that oestrogen receptors (ERs), either associated with or embedded in the plasma membrane, or located in the cytoplasm or the nucleus, coordinate multiple neuroprotective signalling cascades. These include those activated directly by ERs and those activated by the interaction of ERs with the receptors for other neuroprotective factors. Here, we also review these recent discoveries, which represent an essential advance in our understanding of the role of these receptors in the brain and have opened the possibility of using specific ER ligands to promote neuroprotection.

Hormonal oestradiol is neuroprotective

 

Studies in female rodents have shown that decreasing oestradiol levels in plasma by ovariectomy or other means enhances brain damage under neurodegenerative conditions^{6, 7, 8, 9} and decreases brain glucose metabolism and increases amyloid-β oligomers in a mouse model of Alzheimer's disease¹⁰. In turn, the replacement of oestradiol levels in the plasma of ovariectomized animals by hormonal therapy reduces damage after brain injury⁶ and normalizes brain glucose metabolism and decreases amyloid-β oligomers in Alzheimer's disease model mice¹⁰. Furthermore, the magnitude of brain damage after an acute neurotoxic or neurodegenerative insult is influenced by the changes in oestradiol levels in plasma during the oestrous cycle. For example, the administration of the neurotoxic amino acid kainic acid in the morning of oestrus, after the peak of oestradiol in plasma in the afternoon of pro-oestrus, results in less neuronal damage in the hippocampus than the administration of the toxin in the morning of pro-oestrus, before the oestradiol peak⁶.

 

In humans, the decline in oestradiol levels with menopause is associated with an increased risk of cognitive impairment, affective disorders and even Alzheimer's disease pathology¹¹. This suggests that hormonal oestradiol is also neuroprotective in our species. Hormonal therapy — usually a mixture of oestrogens and progestins — has been used for many years to treat the symptoms of menopause, including hot flushes and depression. Prospective studies that have analysed the cognitive outcomes of hormonal therapy show that it enhances cognitive skills — an outcome that is indicative of neuroprotection — in postmenopausal women^{12, 13}.

Brain oestradiol is neuroprotective

 

An important indication of the role of brain-derived oestradiol in neuroprotection was provided by the observation that, in rodents and birds, the brain responds to an acute injury by increasing the expression and activity of aromatase. This was first observed in male and female rodents after an excitotoxic injury in the hippocampus and after stab wound injuries in the cerebral cortex, the hippocampus and other brain regions¹⁴. Both types of brain injury resulted in increased aromatase activity in the injured tissue and de novo expression of aromatase in astrocytes, which do not constitutively express the enzyme in adult rodents. These observations were confirmed in birds, which were shown to express aromatase in radial glia and astrocytes after brain injury¹⁵, and were also extended to other forms of acute brain pathology in rodents, such as experimental stroke¹⁶, global ischaemia-reperfusion¹⁷ and intracranial pressure raising¹⁸. In addition, aromatase expression has been detected in a sexually dimorphic pattern in endothelial cells after brain injury¹⁹.

 

Several experimental approaches have been designed to investigate the function of increased brain aromatase expression and activity after brain injury (Fig. 1). The use of aromatase-knockout (ArKO) mice and the systemic administration of aromatase inhibitors have demonstrated that aromatase is neuroprotective^{7, 8, 9, 20, 21, 22}. However, these models were not able to discriminate between brain aromatase and peripheral aromatase activity. More direct proof of a role for brain-derived oestradiol in neuroprotection was provided by experiments in which brain oestradiol synthesis was reduced through intracerebral administration of aromatase inhibitors. This resulted in decreased brain oestradiol levels and in increased neural damage after the application of different neurodegenerative stimuli in the brains of birds and mammals^{7, 23}. More recently, aromatase antisense oligonucleotides have been used to block aromatase expression and oestradiol synthesis in the rodent hippocampus¹⁷. Aromatase silencing with this method enhanced the effect of global cerebral ischaemia on neuronal loss and gliosis in hippocampal area CA1. All these findings suggest that the brain enhances local oestradiol synthesis after injury as an endogenous neuroprotective mechanism.

 

Figure 1: Neuroprotective actions of brain aromatase

a | Under normal conditions, astrocytes in wild-type (WT) mice do not express the oestradiol-synthesizing enzyme aromatase. b | After an acute brain injury, reactive gliosis (including the conversion of surveillance microglia to reactive microglia) and neuron death occur. However, in the WT mouse brain injury also causes astrocytes to become reactive and express aromatase. Aromatase expression is also observed in neurons and endothelial cells. The increased oestradiol levels resulting from the enhanced expression and activity of aromatase protect neurons and reduce reactive gliosis, as shown on the right of the panel.c | Neuronal degeneration and reactive gliosis, with the subsequent release of pro-inflammatory mediators by astrocytes and microglia, is enhanced after the pharmacological inhibition of brain aromatase or in aromatase-knockout (ArKO) mice.

 An important point to consider is that brain-derived oestradiol is neuroprotective in both male and female animals. In males, aromatase may use circulating testosterone as a precursor to generate oestradiol. Interestingly, electron microscope studies have shown aromatase immunoreactivity in astroglial processes in contact with the basal laminae of capillaries in the injured brain¹⁴. This suggests that as soon as testosterone crosses the blood–brain barrier it can be converted to oestradiol in the astrocyte end-feet. In addition, both male and female rodent brains express the enzymes for steroidogenesis and can produce testosterone from cholesterol (Box 1). Thus, the actual oestradiol levels in the injured brain will depend on the amount of circulating testosterone (predominantly in males), the amount of circulating oestradiol (predominantly in females, but with cyclical changes) and the steroidogenic activity of the brain, which may fluctuate according to different life conditions and is different in males and females²⁴.

 

The importance of aromatase as a neuroprotective molecule in humans is suggested by the existence of genetic variants of the enzyme that confer an increased risk for Alzheimer's disease^{25, 26, 27, 28}. These genetic variants of aromatase may result in decreased oestradiol synthesis in the brain, which, together with decreased serum oestradiol levels in postmenopausal women or serum testosterone levels in aged men, may increase the risk for developing neurodegenerative diseases. In this regard, it is of interest that aromatase expression is increased in astrocytes in the human prefrontal cortex in the late stages of Alzheimer's disease, a phenomenon that has been interpreted to be part of a rescue programme²⁹.

Oestradiol-mediated neuroprotective signaling

 

Role of oestrogen receptors. As in other tissues, oestradiol acts in the brain by activating several complementary signalling mechanisms, the best characterized of which is the regulation of gene transcription through classical intracellular ERs (Fig. 2). Classical ERs are transcription factors that have the peculiarity of being activated by a ligand (oestradiol). At present, two main forms of classical ERs — ERα and ERβ — have been identified and cloned^{30, 31, 32}. Both receptor forms have a similar structure, with a DNA-binding domain and a ligand-binding domain. Oestradiol binds to the ligand-binding domain and induces the activation and the homodimerization or heterodimerization of the ER. Subsequently, the ER binds to oestrogen-responsive elements (EREs) in the promoter region of specific genes through the DNA-binding domain, resulting in the recruitment of transcriptional co-activators and co-repressors³³. Classical ERs may also regulate gene transcription by acting as transcriptional partners at non-ERE sites, such as activating protein 1 (AP1) sites³⁴. In addition, classical ERs have two activation domains, which allow them to be regulated by kinases activated by the signalling pathways of several growth factors, such as insulin-like growth factor 1 (IGF1)^{35, 36}.

 

Figure 2: Oestradiol activates multiple neuroprotective signalling mechanisms.

Oestradiol binds to various oestrogen receptors (ERs). The classical ERs (ERα and ERβ) are transcription factors that, after oestradiol binding, form homodimers or heterodimers and regulate transcription. ERα and ERβ are also associated with the plasma membrane, where they activate different signalling cascades. Oestradiol also activates neuroprotective signalling by binding to other membrane receptors, such as G protein-coupled ER (GPER) and Gαq protein-coupled membrane ER (G_q-mER). In addition, oestradiol promotes neuroprotection by an indirect regulation of the signalling of other receptors, such as insulin-like growth factor 1 (IGF1) receptor (IGF1R), the brain-derived neurotrophic factor (BDNF) receptor TRKB, Notch — a receptor involved in cell-to-cell communication that is activated by membrane ligands in adjacent cells, such as Delta (DLL) and Jagged (JAG) — and the WNT receptor complex, which is composed of Frizzled and lipoprotein receptor-related protein (LRP). The mechanisms by which oestradiol regulates IGF1R, TRKB, Notch and WNT are shown in Fig. 4.

Classical ERs are also associated with plasma membrane lipid rafts³⁷, where they interact with neurotransmitter and growth factor receptor signalling (Fig. 2). Thus, classical ERs are a point of convergence for the signalling of oestradiol and the signalling of other neuroprotective factors. This is an important point to keep in mind when considering the neuroprotective mechanisms of oestradiol and ERs. Finally, oestradiol may bind to membrane-associated non-classical ERs, such as G protein-coupled ER (GPER), a member of the G protein-coupled receptor superfamily, which regulates the activity of extracellular signal-regulated kinases (ERKs) and the phosphoinositide 3-kinase (PI3K) signalling pathway, also allowing the interaction with the signalling of other neuroprotective molecules³⁸. Another membrane-associated non-classical ER is Gαq protein-coupled membrane ER (G_q-mER), which was originally identified in hypothalamic neurons, in which it modulates μ-opioid and GABA neurotransmission³⁹.

 

Both classical and non-classical ERs participate in the neuroprotective actions of oestradiol. Thus, it has been shown that the neuroprotective actions of oestradiol in vivo and in vitro are imitated by selective agonists of ERα, ERβ, GPER and G_q-mER^{40, 41, 42, 43, 44, 45} and are blocked by ER antagonists, ER silencing and ER knockdown^{45, 46, 47}. It is unclear whether all the ERs participate in the protective actions of the molecule in all models of neuronal degeneration. Depending on the pathological model, some receptors seem to have more relevance than others⁴⁸. For example, ERα, but not ERβ, was shown to be involved in the neuroprotective action of oestradiol in a focal ischaemia model, after middle cerebral artery occlusion in mice⁴⁹. However, both ERα and ERβ participate in the induction of neurogenesis by oestradiol after focal ischaemia⁵⁰ and in the neuroprotective action of the hormone after global cerebral ischaemia in rodents^{40, 51}. Also, different receptors seem to activate complementary and often redundant protective signalling pathways to produce the final neuroprotective effect (Fig. 3). 

Figure 3: Redundant neuroprotective signalling elicited by oestrogen receptors.

Oestradiol enhances the expression of anti-apoptotic genes and neuroprotective growth factors and represses the expression of pro-apoptotic genes and pro-inflammatory molecules in the brain. This action of the hormone is mediated by two redundant mechanisms: the binding to the intracellular oestrogen receptors (ERs) ERα and ERβ and subsequent regulation of ER-mediated transcription, and the binding to membrane receptors (ERα, ERβ and G protein-coupled ER (GPER)) and subsequent regulation of kinase-activated transcription factors by the activation of phosphoinositide 3-kinase (PI3K)–AKT, extracellular signal-regulated kinase 1 (ERK1)–ERK2, and Janus kinase (JAK)–signal transducer and activator of transcription 3 (STAT3) signaling.

 The role of ERs in the activation of neuroprotective mechanisms has led researchers to assess the neuroprotective potency of different ER ligands, such as selective ER modulators (SERMs)^{52, 53, 54}. SERMs are clinically used synthetic ER ligands that have some advantages over oestradiol as neuroprotective molecules. The use of oestradiol as a neuroprotectant is limited by its feminizing effects and by its possible role in ER-positive cancers. Some SERMs are free of these limitations and are promising drugs for neuroprotection (Box 3).

Box 3: Selective oestrogen receptor modulators  

Selective oestrogen receptor (ER) modulators (SERMs) bind to the ligand-binding domain of classical ERs and may act as ER agonists or ER antagonists, depending on the tissue. Thus, SERMs such as tamoxifen and raloxifene are used as ER antagonists for the treatment of ER-positive breast cancer, whereas raloxifene and bazedoxifene are used as ER agonists for the prevention of osteoporosis in postmenopausal women and for the treatment of menopausal symptoms, respectively¹⁶⁴. Different SERMs confer a different ER three-dimensional structure, allowing or impeding the recruitment of transcriptional co-activators and co-repressors. This, together with the specific patterns of expression of such transcriptional co-regulators in different cell types, is the cause of the tissue-dependent agonistic or antagonistic activity of SERMs¹⁶⁴. In addition, some SERMs, such as tamoxifen and raloxifene, are agonists of G protein-coupled ER (GPER)¹⁶⁵.

 

The discovery of the role of ERs as coordinators of neuroprotective signalling mechanisms has prompted the investigation of the potential neuroprotective activity of SERMs in various experimental models of brain pathology, including cognitive decline¹⁶⁶, affective disorders^{167, 168}, traumatic brain and spinal cord injury^{169, 170, 171}, subarachnoid haemorrhage¹⁷², stroke^{173, 174, 175}, neuroinflammation¹⁷⁶ multiple sclerosis^{177, 178}, Alzheimer's disease^{179, 180} and Parkinson's disease¹⁸¹. These studies, together with some clinical findings¹⁸², support the possible use of SERMs as new drugs for the treatment of acute and chronic neurodegenerative diseases, affective disorders and cognitive decline.

 

Cell types involved. Oestradiol-mediated neuroprotective signalling mechanisms are activated in various cell types in the brain. Many in vitro studies have shown that oestradiol promotes neuronal survival in primary neurons in the absence of glial cells and that oestradiol directly activates survival mechanisms in neurons^{55, 56, 57}. Neurons constitutively express aromatase and therefore are a source of neuroprotective oestradiol. Indeed, oestradiol produced by neurons in vitro is neuroprotective⁵⁸. In addition, some studies suggest that neuronal production of oestradiol may increase under neurodegenerative conditions. For example, it is known that excitotoxic brain injury increases the expression of steroidogenic acute regulatory protein (StAR) in hippocampal neurons, suggesting increased neuronal steroidogenesis under neurodegenerative conditions⁵⁹ (see Box 1 for the role of StAR in steroidogenesis). In addition, sciatic nerve chronic constriction injury increases aromatase activity and oestradiol formation in dorsal root ganglion neurons⁶⁰ but not in satellite glial cells. Furthermore, increased aromatase expression has been detected in hippocampal neurons of spontaneously hypertensive rats⁶¹. Interestingly, oestradiol increases aromatase expression in hippocampal neurons of hypertensive rats⁶¹. This may represent a positive-feedback mechanism in which the production of oestradiol by neurons further enhances the expression of aromatase and the synthesis of oestradiol in this cell population. Neuronal oestradiol may act as an autocrine or paracrine factor to increase neuronal survival acting on neurons expressing ERs. In this regard, it is of interest that ERα is upregulated in neurons after brain ischaemia and that oestradiol potentiates this upregulation⁴⁶. In addition, aromatase inhibition results in the downregulation of ERα and the upregulation of ERβ in hippocampal neurons⁶². Thus, we may envisage a mechanism by which oestradiol, generated by neurons in response to brain injury, enhances further oestradiol production in these cells and at the same time increases oestradiol-mediated neuroprotective signalling by upregulating ERα in neurons.

 

Although oestradiol may directly act on neurons to promote neuroprotection in vitro, the participation of other cell types is also necessary to maintain global tissue homeostasis in vivo^{43, 63}. Thus, oestradiol acts on glial and endothelial cells to maintain the function of the neurovascular unit, regulates the inflammatory response of astrocytes and microglia to control neuroinflammation and acts on neurons, astrocytes and oligodendrocytes to maintain the function and propagating properties of neuronal circuits. As mentioned above, the main function of oestradiol as a hormonal signal is to maintain the homeostatic equilibrium of the organism, and this function of oestradiol is also reflected under pathological conditions at the tissue level.

 

The role of non-neuronal cells in the neuroprotective properties of oestradiol has been recently reviewed^{63, 64, 65} and is not examined here in detail. Our main focus in this Review is the molecular mechanisms elicited by oestradiol in the brain in vivo, in which the cell types involved have not always been identified. However, it should be noted that glial cells express ERs, including ERα, ERβ and GPER^{64, 66, 67}, and that brain injury induces both the synthesis of oestradiol in reactive astrocytes and the expression of ERs in these cells⁶⁸. This suggests that astrocytes may play an important part in the neuroprotective actions of oestradiol. Indeed, recent studies using conditional knockout mice deficient in either ERα or ERβ have shown that, in an experimental model of multiple sclerosis, the protective action of oestradiol is mediated by ERα expressed in astrocytes but not by ERα expressed in neurons or ERβ expressed in astrocytes or neurons⁴³.

 

ERs in glial cells activate several neuroprotective mechanisms in response to oestradiol, including the release of factors that have trophic effects on neurons and other cell types and the control of neuroinflammation, oedema and extracellular glutamate levels. Classical ERs associated with the plasma membrane of astrocytes are involved in the oestradiol-induced release of transforming growth factor-β (TGFβ) through the activation of the PI3K–AKT signalling pathway^{69, 70}. In addition, ERα and ERβ are involved in the anti-inflammatory actions of oestradiol on microglia and astrocytes^{71, 72}. The anti-inflammatory action of ERα is also exerted through the activation of the PI3K pathway, which in turns blocks nuclear factor-κB (NFκB) activation and translocation to the cell nucleus⁷³. ERβ also has an essential role in the regulation of the neuroinflammatory response of astrocytes. This effect is in part mediated by the upregulation of neuroglobin⁷⁴, a haemoprotein that shares partial sequence identity with vertebrate haemoglobin and myoglobin, which protects neurons from a variety of insults, such as hypoxia, glucose deprivation, oxidative stress, β-amyloid toxicity and experimental stroke.

 

Main neuroprotective signalling cascades. ERα, ERβ and GPER trigger parallel neuroprotective mechanisms in the brain (Fig. 3), including the activation of extracellular signal-regulated kinase 1 (ERK1)–ERK2 (also known as MAPK3–MAPK1) and PI3K neuroprotective signalling cascades as well as the inhibition of pro-apoptotic JUN amino-terminal kinase (JNK) signalling^{41, 45, 75}. Studies on primary cortical neurons have shown that oestradiol activates ERK1–ERK2 and PI3K neuroprotective signalling in parallel in the same neurons⁷⁶. The inhibition of either ERK1–ERK2 or PI3K signalling abolishes the neuroprotective actions of oestradiol in various neurodegenerative models, including global cerebral ischaemia and experimental stroke models^{75, 77}.

 

An important component of the neuroprotective mechanisms of oestradiol is the regulation of B cell lymphoma 2 (BCL-2) family members that are involved in the control of apoptosis (Fig. 3). In the brain, oestradiol upregulates the expression of anti-apoptotic BCL-2 family members, such as BCL-2 (Refs 78,79,80,81,82), BCL-XL^{83, 84, 85} and BCL-W⁸⁶, and downregulates the expression of pro-apoptotic BCL-2 family members, such as BCL-associated death promoter (BAD)⁸⁷ and BCL-2-interacting mediator of cell death (BIM)⁸⁶. Both ERα and ERβ mediate the oestradiol-induced increase in the expression of BCL-2 in hippocampal neurons⁸⁸. The activation of either the ERK1–ERK2 or the PI3K signalling pathways by ERs results in the decreased expression of BAD and in the upregulation of BCL-2 (Refs 89,90,91,92). ER activation also induces the transcription of BCL2 and another anti-apoptotic gene, survivin, through signal transducer and activator of transcription 3 (STAT3), a transcription factor that mediates neuroprotective actions of the hormone in experimental cerebral ischaemia^{93, 94, 95}. In addition, some members of the BCL-2 family, such as BCL-W and BIM are regulated by oestradiol through the inhibition of JNK⁸⁶. Thus, several redundant mechanisms are elicited by oestradiol to control apoptosis in the brain.

 

Glycogen synthase kinase 3β (GSK3β) has been proposed to be a potential target for the treatment of neurodegenerative diseases, given its role in neurodegeneration and regeneration processes⁹⁶. One of the effects of the abnormal activation of GSK3β in neurodegenerative diseases is the hyperphosphorylation of the microtubule associated protein Tau, which is the main cause of the dysfunction of this protein in Alzheimer's disease and other tauopathies⁹⁷. The activity of GSK3β is regulated by phosphorylation at different residues. Its activity is decreased by phosphorylation at Ser9 by AKT. The inhibition of GSK3β activity is a common mechanism of neuroprotection by several factors, such as WNT, IGF1 and oestradiol. Oestradiol, via ERα and GPER, activates PI3K in the brain and in primary neurons. PI3K, in turn, phosphorylates and activates AKT^{38, 98, 99}, which phosphorylates and inhibits GSK3β and reduces Tau phosphorylation^{90, 98} (Fig. 4).

Figure 4: Oestradiol mediates indirect regulation of other neuroprotective signalling pathways.

a | Oestradiol (E2) activates insulin-like growth factor 1 (IGF1) receptor (IGF1R)-mediated neuroprotective signalling through the transcriptional upregulation of IGF1 and by inducing the interaction of oestrogen receptor-α (ERα) with the p85 catalytic subunit of phosphoinositide 3-kinase (PI3K, which is composed of p85 and p110 subunits). This allows the formation of a multimolecular complex made up of ERα, IGF1R and components of the IGF1R signalling pathway, such as insulin receptor substrate 1 (IRS1), PI3K, AKT and glycogen synthase kinase 3β (GSK3β). Through this pathway, oestradiol downregulates Tau phosphorylation and induces the stabilization of β-catenin. In addition, in the presence of oestradiol, IGF1 downregulates the transcriptional activity of ERα through the PI3K–AKT–GSK3β–β-catenin signalling pathway. b | Oestradiol activates WNT signalling by inhibiting of the expression of Dickkopf 1 (DKK1), a molecule that binds to lipoprotein receptor-related protein (LRP) and prevents the formation of the signalling complex of WNT with Frizzled and LRP. The inhibition of the expression of DKK1 by oestradiol is mediated by the attenuation of JUN amino-terminal protein kinase (JNK)–JUN signalling through a mechanism involving membrane ERs. The activation of WNT signalling by oestradiol results in the inhibition of GSK3β and the translocation of β-catenin to the cell nucleus, where it regulates transcriptional activity through T cell factor (TCF) and lymphoid enhancer-binding factor (LEF), promoting the expression of pro-survival molecules. c | Oestradiol increases the transcription of brain-derived neurotrophic factor (BDNF), which activates TRKB–PI3K-mediated neuroprotective signalling. d | Oestradiol, through a mechanism involving G protein-coupled ER (GPER) and PI3K, inhibits Notch signalling to the nucleus and promotes neuritogenesis. Note that several signalling pathways activate PI3K-mediated neuroprotective signalling. DLL, Delta; JAG, Jagged.

In addition to reducing Tau phosphorylation, the neuroprotective actions of the inhibition of GSK3β also involve the regulation of β-catenin. The levels of β-catenin are regulated by oestradiol through the ERα–PI3K–AKT–GSK3β signalling pathway^{98, 100}. Studies in neuroblastoma cells have shown that the activation of the PI3K–AKT–GSK3β pathway induces the translocation of β-catenin to the cell nucleus, which inhibits the transcriptional activity of ERα³⁶ and regulates transcriptional activity through T cell factor (TCF) and lymphoid enhancer-binding factor (LEF) proteins¹⁰¹ (Fig. 4). The set of genes regulated by oestradiol through this mechanism is similar but not identical to the set regulated by the canonical WNT signalling pathway¹⁰¹ (see below).

Interaction with other factors

 

As indicated above, the neuroprotective mechanisms of ERs also involve interactions with the protective pathways induced by other neuroprotective factors (Fig. 4).

 

Brain-derived neurotrophic factor. Oestradiol increases the phosphorylation of cyclic AMP-responsive element-binding (CREB) protein, the expression of its target gene brain-derived neurotrophic factor (BDNF) and the phosphorylation of the BDNF receptor TRKB (also known as NTRK2) in the CA1 region of the hippocampus and in other brain regions. This action of oestradiol is mediated, at least in part, through the ERK1–ERK2 and PI3K signalling pathways^{75, 82, 91, 102, 103, 104}. BDNF is a neuroprotective factor in neurodegenerative and affective disorders and contributes to the homeostatic and neuroprotective actions of oestradiol in the hippocampus, cooperating with the actions of the hormone on synaptic plasticity, neurogenesis and cognition^{104, 105, 106, 107}.

 

Insulin-like growth factor 1. Like oestradiol, IGF1 is a neuroprotective hormone and a local factor that contributes to maintain body and tissue homeostasis, including the regulation of brain plasticity and function¹⁰⁸. The neuroprotective actions of oestradiol and IGF1 have many points in common, and both factors interact in the regulation of neuronal survival, neuritogenesis, adult neurogenesis, brain cholesterol homeostasis, glucose metabolism, neuroprotection and cognition^{82, 109, 110, 111, 112, 113, 114, 115}.

 

Oestradiol signalling and IGF1 receptor (IGF1R) signalling interact through ERα (Fig. 4). Oestradiol induces the interaction of ERα with the p85 catalytic subunit of PI3K, probably in specific plasma membrane domains, such as the lipid rafts¹¹⁶. This allows the formation of a multimolecular complex composed of ERα, IGF1R and components of the IGF1R signalling pathway, such as insulin receptor substrate 1 (IRS1), PI3K, AKT and GSK3β^{98, 117}. The interaction of ERα with IGF1R signalling allows the PI3K–AKT–GSK3β–β-catenin signalling pathway to be regulated by oestradiol^{90, 98}. In addition, IGF1 regulates the transcriptional activity of ERα. In the presence of oestradiol, IGF1 downregulates the transcriptional activity of ERα through the PI3K–AKT–GSK3β–β-catenin signalling pathway³⁶ (Fig. 4). By contrast, in the absence of oestradiol, IGF1 is known to enhance the transcriptional activity of ERα in neuroblastoma cells³⁵. This suggests that the interaction of ERα and IGF1R constitutes a coincidence signal detector mechanism that allows the biological response to be adapted in response to changing levels of oestradiol and IGF1 under physiological and pathological conditions. This mechanism may be highly relevant under neurodegenerative conditions, as brain injury is associated with increased local expression of ERs and IGF1Rs, as well as increased levels of local oestradiol and IGF1 (Refs 14,40,46,68,112). In addition, plasma levels of oestradiol, the oestradiol precursors testosterone and dehydroepiandrosterone and IGF1 decrease with ageing in men and women, which may affect the function of the coincidence signal detector mechanism. This may explain why oestradiol is protective against experimental stroke in young female mice but increases brain damage after stroke in aged female mice, in which levels of IGF1 are decreased¹¹². Indeed, oestradiol recovers its neuroprotective actions in aged female mice treated with IGF1 (Ref. 112).

 

WNT. As mentioned above, oestradiol activates β-catenin-mediated transcription through the PI3K–AKT–GSK3β signalling pathway. In addition, oestradiol activates β-catenin-mediated transcription through the canonical WNT signalling pathway, which is also known to be neuroprotective^{118, 119} (Fig. 4). The canonical WNT signalling pathway is activated when WNT binds to its co-receptors, low-density lipoprotein-related protein 5 (LRP5), LRP6 and Frizzled. The activation of canonical WNT signalling results in the stabilization of cytosolic β-catenin and its translocation to the cell nucleus, where it regulates transcriptional activity through TCF–LEF. Oestradiol upregulates WNT signalling by inhibiting the expression of Dickkopf 1 (DKK1)^{120, 121}, a molecule that binds to LRP5–LRP6 and prevents the formation of the signalling complex of WNT with Frizzled and LRP5–LRP6. Inhibition of canonical WNT signalling by DKK1 results in the downregulation of BCL-2 and the upregulation of the pro-apoptotic molecule BCL2-associated protein X (BAX) and the hyperphosphorylation of Tau¹²². DKK1 expression is enhanced in neurodegenerative conditions, such as global cerebral ischaemia^{120, 121}. Experiments have shown that oestradiol prevents the upregulation of DKK1 under these conditions and that the inhibition of DKK1 expression by oestradiol promotes neuronal survival, reduces Tau hyperphosphorylation and reduces ischaemia-induced neuronal damage^{120, 121}. The inhibition of the expression of DKK1 by oestradiol is mediated by the attenuation of JNK–JUN signalling¹²⁰, which is enhanced by global cerebral ischaemia¹²⁰ and is downregulated by oestradiol through AKT¹²³.

 

Notch. Notch signalling is a conserved mechanism involved in direct cell-to-cell communication. In neurons, the inhibition of Notch signalling promotes neuronal differentiation¹²⁴. Notch upregulates the expression of the transcription factor Hairy and enhancer of Split 1 (HES1), which represses the transcription of the neuritogenic factor neurogenin 3 (NGN3; also known as NEUROG3)¹²⁴. Recent studies have shown that oestradiol inhibits Notch signalling in primary neurons, decreasing the expression of HES1 and therefore increasing the expression of NGN3 and neuritogenesis^{38, 124}. The oestrogenic regulation of Notch signalling and NGN3 expression is mediated by GPER and the activation of the PI3K–AKT signalling pathway^{38, 124} (Fig. 4). The regulation of Notch signalling in neurons may be involved in the regenerative actions of the hormone by acting to promote neuritic growth.

Conclusions and future directions

 

The available evidence, reviewed here, suggests an integrated view of oestradiol-mediated neuroprotective signalling. Oestradiol synthesis and ER expression are induced after brain injury, and the newly-synthesized oestradiol activates multiple neuroprotective signalling mechanisms, limiting neural damage by preventing secondary neurodegeneration²³. The expression of aromatase and the synthesis of oestradiol are also enhanced in the brain under various neurodegenerative conditions. De novo induction of aromatase in reactive astrocytes has been detected in conditions of acute neurodegeneration, such as physical brain injury^{14, 15}, excitotoxicity¹⁴, global cerebral ischaemia²⁰ and stroke^{16, 22}. By contrast, in the two models of chronic neurodegeneration in which brain aromatase has been examined — hypertension⁶¹ and sciatic nerve chronic constriction injury⁶⁰ — the expression of the enzyme is enhanced in neurons and not in astrocytes. However, aromatase expression is increased in astrocytes in the human prefrontal cortex in the late stages of Alzheimer's disease²⁹. Thus, at least in some cases, aromatase expression is induced in astrocytes under chronic neurodegenerative conditions. The factors that induce the expression of aromatase in neurons and astrocytes after brain injury remain to be identified¹²⁵.

 

Neurodegenerative conditions also result in an enhanced expression of ERα in neurons and in increased expression of ERα and ERβ in astrocytes. Thus, oestradiol synthesized by brain aromatase may activate ERα-associated neuroprotective signalling pathways in neurons and activate ERα and ERβ signalling in astrocytes to modulate neuroinflammation, oedema and the release of neuroprotective factors. In addition, locally produced oestradiol may also target ERs in other cell types, such as microglia, oligodendrocytes or endothelial cells. Further studies should determine whether neurodegenerative conditions regulate other ERs that mediate neuroprotective actions of oestradiol, such as GPER or G_q-mER.

 

Interestingly, oestradiol exerts a positive-feedback action on the expression of brain aromatase and ERα in neurons. Thus, aromatase induction after brain injury seems to initiate a cycle that potentiates brain oestradiol synthesis and oestradiol-mediated neuroprotective signalling, reducing neurodegeneration. Furthermore, recent studies suggest that brain oestradiol synthesis not only is an endogenous mechanism of neuroprotection but is also essential for the neuroprotective action of hormonal oestradiol or oestradiol therapies. Thus, in H19-7 hippocampal cells, the inhibition of aromatase prevents the neuroprotective action of exogenous oestradiol¹²⁶. In addition, the neuroprotective action of circulating oestradiol in a mouse model of Alzheimer's disease is significantly reduced in ArKO mice that have undetectable levels of brain oestradiol¹²⁷. Further studies are necessary to clarify the role of brain oestradiol synthesis in the neuroprotective outcome of hormone therapy.

 

The studies reviewed here suggest that brain levels of oestradiol are highly critical for neuroprotection. Unfortunately, our knowledge on brain oestradiol levels — which can only be adequately measured in tissue samples — in humans is very poor. New positron emission tomography imaging techniques for the localization of aromatase in the human living brain¹²⁸ may help us to better understand the role of the enzyme and that of brain-derived oestradiol in neurologic, affective and cognitive alterations in men and women.

 

In this Review, we emphasized the multiple and parallel neuroprotective signalling cascades that are activated by oestradiol in the brain. This neuroprotective signalling is initiated, in part, by several membrane-associated ERs and involves the regulation of the activity of different kinases that finally regulate transcriptional activity. In parallel, intracellular ERs directly or indirectly regulate gene expression, contributing to neuroprotection. There is partial redundancy in the neuroprotective actions activated by different ERs, as some of them activate similar protective mechanisms, modulating the expression of the same neuroprotective growth factors, inflammatory mediators and apoptosis regulators. In addition, both membrane-associated and intracellular ERs contribute to the interaction of oestradiol with the neuroprotective signalling of other factors, such as BDNF⁷⁵, IGF1 (Ref. 36), WNT¹²¹ and Notch¹²⁴. There is also partial redundancy but not complete overlap in the neuroprotective signalling that is directly activated by ERs and the neuroprotective signalling that is activated by ERs through other factors.

 

Although oestradiol activates multiple neuroprotective signalling pathways, several studies have shown that the inhibition of individual pathways can dramatically reduce or abolish neuroprotection. This may indicate that certain signalling pathways are more relevant than others depending on the type of neurodegenerative condition. Indeed, the signalling of some ERs seems to be more relevant than that of other ERs, depending on the pathological model and the readout examined. However, we favour the idea that the global homeostatic neuroprotective action of oestradiol may be achieved only by the coordinated action of multiple mechanisms, all of them indispensable. Thus, inhibition of an individual mechanism may compromise the final neuroprotective outcome. In this regard, an important gap in our knowledge is how these multiple mechanisms are coordinated in time and among the different brain cells. The limited evidence available indicates that both neurons and glial cells participate in the protective actions of the hormone in vivo. However, it is unclear how neuron-initiated and glia-initiated neuroprotective signalling are coordinated. The potential role of endothelial cells in the neuroprotective actions of oestradiol, in particular after stroke¹⁹, also needs further investigation.

 

Another major gap in our knowledge on the neuroprotective signalling mechanisms of oestradiol is the role of sex differences. Neuroprotective oestradiol signalling has been analysed in male and female animals, but a systematic approach has been rarely performed. Thus, sex is an important factor that should be considered in future studies, given the reported sex differences in the onset, manifestation and outcome of different neurodegenerative diseases and affective disorders, as well as in the response of neural tissue to oestradiol^{129, 130, 131}.

 

Age is also an important factor to take into consideration in future research on neuroprotective oestradiol signalling, as ageing per se, or age after menopause, is known to affect the response of the brain to the hormone. Animal studies^{121, 132, 133, 134} and clinical observations^{11, 135, 136} suggest that hormonal therapy has a window of opportunity in the perimenopausal period during which it may reduce cognitive decline. Several studies have addressed the question on whether age or prolonged ovarian hormone deprivation affect the neuroprotective actions of oestradiol. For example, the neuroprotective action of the hormone on experimental stroke in young female mice is impaired in aged female mice, probably because the decrease in IGF1 plasma levels with ageing alters the cross-talk between ERα and IGF1R¹¹². However, high physiological doses of oestradiol are able to protect the brain of aged rats from global cerebral ischaemia¹³⁷. Further studies are therefore necessary to determine in more detail how ageing affects the neuroprotective signalling mechanisms elicited by oestradiol.

 

Most of the neuroprotective oestrogen signalling mechanisms reviewed here have been characterized in acute models of neurodegeneration, such as experimental stroke, global cerebral ischaemia or acute brain injury. The information obtained with these models may not be directly applicable to chronic neurodegenerative diseases, in which neuronal degeneration and death is a slow process accompanied by chronic neuroinflammation. Further studies are necessary to determine the long-term protective signalling mechanisms of oestradiol in chronic neurodegenerative diseases and to analyse the neuroreparative potency of oestradiol, which has been poorly explored. One promising research direction is to determine the role of metabolic control on the neuroprotective actions of oestradiol, as the hormone may exert sustained protective and reparative actions by maintaining metabolic homeostasis in the brain¹¹⁵. Acting on mitochondria, oestradiol regulates glucose metabolism, glycolysis and the tricarboxylic acid cycle-coupled oxidative phosphorylation and ATP generation in neurons¹¹⁵. In addition, the hormone regulates whole-body metabolism by acting on adipose tissue, the pancreas and the hypothalamus^{115, 138}. Substantial evidence supports an influence of metabolic alterations on chronic neurodegenerative diseases and cognitive impairment. Therefore, further studies should determine whether the metabolic control exerted by oestradiol is a principal component of its long-term neuroprotective actions.

 

Further research should also explore alternatives to oestradiol therapy. In addition to new SERMs that may activate the multiple neuroprotective mechanisms of action of ERs, another promising therapeutic avenue for brain diseases is the development of selective neuroprotective ERα, ERβ and GPER ligands^{139, 140}. In addition, as local oestradiol synthesis in the brain is neuroprotective, studies should be directed to understand the mechanisms that regulate aromatase expression in the brain. The human aromatase gene promoter region is highly complex, with promoters that are used with tissue specificity¹⁴¹. Thus, aromatase tissue-specific regulation by natural or synthetic compounds opens the possibility for selective modulation of oestrogen biosynthesis within the human brain¹⁴².

Acknowledgements

The authors thank M. Garcia-Diaz, Stony Brook University School of Medicine, New York, USA, for critical reading of the manuscript. The authors acknowledge support from Ministerio de Economía y Competitividad, Spain (BFU2011-30217-C03-01).

Author information

 Maria-Angeles Arevalo,^1, Iñigo Azcoitia^2, & Luis M. Garcia-Segura^1, 

 

Affiliations

1. Instituto Cajal, Consejo Superior de Investigaciones Científicas, E-28002 Madrid, Spain.

2. Department of Cell Biology, Faculty of Biology, Universidad Complutense, E-28040 Madrid, Spain.

Competing interests statement

The authors declare no competing interests.

Corresponding author

Correspondence to:

• Luis M. Garcia-Segura

Author details

• Maria-Angeles Arevalo

  Maria-Angeles Arevalo obtained her Ph.D. in chemistry from the Autonomous University of Madrid, Spain. She worked as a postdoctoral fellow at the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid. She is a staff scientist at the Cajal Institute in Madrid, where she co-chairs the Laboratory of Neuroactive Steroids. Her research focuses on the molecular mechanisms mediating the actions of oestradiol on neurons and glial cells. Maria-Angeles Arevalo's homepage.

• Iñigo Azcoitia

  Iñigo Azcoitia obtained his Ph.D. in biology from the Complutense University of Madrid. He is a professor in the Department of Cell Biology at the Complutense University School of Biology. His research focuses on the neuroprotective mechanisms of sex steroids and on their role in the regulation of adult neurogenesis.

• Luis M. Garcia-Segura

  Luis M. Garcia-Segura obtained his Ph.D. in biology from the Complutense University of Madrid. He worked at Geneva University School of Medicine, Switzerland, Yale University School of Medicine, New Haven, Connecticut, USA, and The Babraham Institute, Cambridge, UK. He is a research professor at the Cajal Institute, where he co-chairs the Laboratory of Neuroactive Steroids. His research focuses on the neuroprotective mechanisms of neuroactive steroids. Luis M. Garcia-Segura's homepage.

 

 Nature Reviews Neuroscience | Review

Nature Reviews Neuroscience (2014)

DOI: doi:10.1038/nrn3856

Published online 26 November 2014

 

 

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Enabling the 'host jump': structural determinants of receptor-binding specificity in influenza A viruses

The shift in the receptor-binding specificity of influenza A viruses is mostly determined by mutations in viral haemagglutinin. In this Review, Gao and colleagues discuss recent crystallographic studies that provide molecular insights into haemagglutinin–host receptor interactions that have enabled several influenza A virus subtypes to ‘jump’ from avian to human hosts.

Abstract

The recent emergence of the H7N9 avian influenza A virus and its ability to infect humans emphasize the epidemic and pandemic potential of these viruses. Interspecies transmission is the result of many factors, which ultimately lead to a change in the host tropism of the virus. One of the key factors involved is a shift in the receptor-binding specificity of the virus, which is mostly determined by mutations in the viral haemagglutinin (HA). In this Review, we discuss recent crystallographic studies that provide molecular insights into HA–host receptor interactions that have enabled several influenza A virus subtypes to 'jump' from avian to human hosts.

Key points

• Interspecies transmission of influenza A viruses is the result of many factors. One of the key factors involved is a shift in the receptor-binding specificity of the virus, which is mostly determined by mutations in viral haemagglutinin (HA).
• Recent structural studies have provided molecular insights into the HA–host receptor interactions that have enabled several influenza A virus subtypes to jump from avian to human hosts.
• The combination of distinct amino acids at positions 225 and 190 of HA is important for determining the receptor-binding specificity of the H1 subtype.
• The Q226L and G228S substitutions in the HA glycoproteins of the H2 and H3 subtypes are sufficient to change the binding preference from the avian receptor to the human receptor.
• In an experimentally adapted H5 subtype, the Q226L substitution and loss of a glycosylation site near the receptor-binding site contribute to the shift in binding preference from the avian receptor to the human receptor.
• In the H7 subtype, amino acid substitutions at positions 186 and 226 of HA increase the preference for binding to the human receptor.

Introduction

Influenza outbreaks have occurred since at least the Middle Ages, if not since ancient times¹. In the past century, there were four severe influenza pandemics, in 1918 (Spanish flu), in 1957 (Asian flu), in 1968 (Hong Kong flu) and in 2009 (swine flu), as well as a moderate pandemic in 1977 (Russian flu)¹. There is a high probability that we will face another influenza pandemic, but it is impossible to predict when it will happen, where it will originate, what virus subtype will cause the pandemic and the severity of such an outbreak. However, it is likely that new animal-derived influenza strains, particularly avian strains, will contribute to new pandemics², and an increased understanding of the molecular mechanisms involved in determining influenza host tropism should facilitate such predictions in the future.

 

Influenza A virus is a zoonotic pathogen that can infect a broad range of species, including birds, pigs, dogs, horses, tigers and humans, causing annual epidemics (known as seasonal flu) and, at irregular intervals, pandemics (Box 1). The virus is an enveloped, single-stranded, negative-sense RNA virus with a segmented genome comprising eight gene segments³ that encode 16 proteins^{4, 5} (Fig. 1a), although not all viruses express all 16 proteins. Haemagglutinin (HA) and neuraminidase (NA) are the two major viral envelope glycoproteins that recognize sialic acid (SA) on host cells. HA binds to sialylated host cell receptors and mediates membrane fusion, whereas NA removes sialyl residues from the membrane of infected cells and from viral membranes to enable budding and release of newly synthesized virus particles⁶. In the infected host, both HA and NA are targeted by neutralizing antibodies, and based on their antigenic properties, influenza type A viruses are classified into 18 HA subtypes (H1–H16 in wild waterfowl, and H17 and H18 in bats; note that the functions of the bat HA subtypes are currently unknown) and into 11 NA subtypes (N1–N9 in wild waterfowl, and N10 and N11 in bats)^{6, 7, 8, 9, 10}.

 

Figure 1: Structure and life cycle of influenza A viruses.

a | Influenza A viruses are enveloped, single-stranded, negative-sense RNA viruses that contain eight gene segments that encode 16 proteins (although not all influenza viruses express all 16 proteins). The non-structural segment encodes the nuclear export protein NS2 and the host antiviral response antagonist NS1; the matrix segment encodes the matrix protein M1, the ion channel protein M2 and the M2-related protein M42 (which can functionally replace M2); the haemagglutinin (HA) segment encodes the receptor-binding glycoprotein HA; and the neuraminidase (NA) segment encodes NA (which cleaves sialic acid from cell surfaces). In addition, nucleoprotein (NP) and the components of the RNA-dependent RNA polymerase complex (PB1, PB2 and PA) are expressed from their respective genome segments. The two newly identified proteins N40 (the function of which is unknown⁹³) and PA-X⁹⁴, which represses cellular gene expression, are encoded by the PB1 and PA segments, respectively. Another two forms of PA (with amino-terminal truncations) have been found recently, named PA-N155 and PA-N182, which are likely to have important functions in the replication cycle of influenza A viruses⁵. In addition, some viruses express the pro-apoptotic protein PB1-F2, which is encoded by a second ORF in the PB1 segment. b | Virus infection is initiated by binding of the virus to sialylated host cell-surface receptors, and entry is mediated by endocytosis. In the host cell, fusion of viral and endosomal membranes occurs at low pH, which enables the release of the segmented viral genome into the cytoplasm. The viral genome is subsequently translocated to the nucleus, where it is transcribed and replicated. Following synthesis in the cytoplasm, viral proteins are assembled into viral ribonucleoproteins (vRNPs) in the nucleus. Export of vRNPs to the cytoplasm is mediated by M1 and NS2. Virus particles are assembled at the cell membrane, and the newly generated progeny virus buds into extracellular fluid.

To achieve interspecies transmission (known as a 'host jump'), influenza A virus must change its tropism to preferentially target new host species, and both viral and host factors have been implicated in this event^{11, 12}. The high mutation rate of the virus enables it to evolve rapidly and thereby overcome host barriers. All eight gene segments evolve continuously, but this evolution is most pronounced for the HA and NA glycoproteins. Evolution is achieved by two main mechanisms: genetic reassortment between different subtypes (known as antigenic shift if it occurs in either the HA or NA segments) and point mutations owing to antibody-mediated immune pressure (known as antigenic drift), including substitutions, deletions and insertions within the antibody-binding sites. This results in the generation of modified influenza virus genomes, which facilitates virus evasion of the host immune response.

 

HA proteins exhibit specific binding affinities for the different SA-linked glycoproteins that are expressed on cell-surface receptors. Avian viruses preferentially bind to SA linked to the terminal oligosaccharide by an α2,3 bond (which is referred to as the avian receptor), whereas human strains favour the α2,6-linked SA receptor (which is referred to as the human receptor). Specific amino acid mutations in HA lead to a change in receptor-binding preference and thus to altered host specificity and tropism. In addition to the structural determinants of viral HA and their corresponding receptors, other viral determinants contribute to host-specific adaptation, such as the balance between HA receptor-binding activity and NA-mediated release from infected cells, and amino acid substitutions in viral RNA polymerase (reviewed in Refs 11,12).

 

In this Review, we describe recent crystallographic studies that have identified the structural determinants of viral HA that enable interspecies transmission, and we also briefly consider corresponding changes in the host receptor. First, we provide a brief overview of the host cell receptors and viral HA proteins that are involved in the initial stages of influenza A virus infection. We then discuss recent crystallographic structures of the H1, H2, H3, H5 and H7 HA subtypes in complex with both avian and human receptors, with a focus on the amino acid substitutions in the receptor-binding site of HA that enable the host jump. Please note, all amino acid residues throughout the paper are numbered according to the H3 subtype (as is convention in the field), which enables the different virus subtypes to be compared.

 

Figure 2: The haemagglutinin binding site and host receptors.

a | The crystal structure of the ectodomain of haemagglutinin (HA) reveals two distinct domains: the globular domain and the stem domain. The receptor-binding site is located on the membrane-distal globular domain and forms a shallow pocket comprising three secondary elements: the 130-loop, 190-helix and 220-loop (the numbers correspond to the amino acids in the H3 subtype; see inset). Four highly conserved residues (Y98, W153, H183 and Y195) form the base element of the receptor-binding site (indicated in orange). The fusion peptide, which inserts into the host membrane during membrane fusion, is indicated. The structural figures were created using Protein Data Bank (PDB) accession 4JUG. b,c | The HA receptor analogues can be categorized into two types: the avian receptor analogue (α2,3-linked sialylated glycan receptor) (part b) and the human receptor analogue (α2,6-linked sialylated glycan receptor) (part c). Sialylated glycans of the host receptors that influenza viruses bind to contain the three terminal saccharides: the terminal sialic acid SA1, the galactose ring Gal2 (at position two relative to SA) and N-acetylglucosamine GlcNAc3 (at position three relative to SA). When bound by HA, the α2,3-linked SA receptor adopts a trans conformation and the hydrophilic glycosidic oxygen atom faces the 220-loop in the receptor-binding site (part b), whereas the α2,6-linked SA receptor adopts a cis conformation and the hydrophobic C6 atom points towards the 220-loop (part c). The structural figures are created using the PDB accessions 4JUH and 4JUJ.

Figure 3: Haemagglutinin proteins from the H1, H2 and H3 subtypes in complex with the avian and human receptor analogues.

Crystal structures of H1, H2 and H3 haemagglutinin (HA)–receptor complexes have revealed the structural basis for the switch in binding specificity. a | H1 HA from the avian subtype contains the residues E190 and G225 in the receptor-binding site, and can bind to both α2,3-linked (avian; shown in cyan) and α2,6-linked (human; shown in magenta) sialic acid (SA) receptors. The structural figures were created using Protein Data Bank (PDB) accessions 1RVX and 1RVZ. b | H1 HA proteins from two human isolates from the 1918 and 2009 pandemics contain the residues D190 and D225 and specifically bind to the human receptor. The crystal structure revealed that D190 interacts with N-acetylgalactosamine GlcNAc3, whereas D225 contacts the galactose ring Gal2.These interactions are absent in the avian HA–receptor complex owing to the different configurations of the human receptor (folded) compared with the avian receptor (extended)^{29, 30}. The structural figure was created using the PDB accession 4JTV. c | H1 HA mutants from later isolates from the 1918 and 2009 pandemics contain the residues D190 and G225 and can bind to both avian and human receptors. It is likely that G225 increases the flexibility of the 220-loop, which provides a suitable microenvironment to accommodate the extended configuration of the avian α2,3-linked SA receptor. The structural figures were created using PDB accessions 4JUH and 4JUJ. d | Avian HA proteins from the H2 and H3 subtypes contain the residues Q226 and G228, and can bind to both avian and human receptors. The structural figures were created using PDB accessions 2WR3 and 2WR4. e | HA proteins from human-adapted H2 and H3 subtypes contain the residues L226 and S228, and preferentially bind to the human receptor. L226 creates a hydrophobic environment that is favourable for the orientation of the hydrophobic C6 atom of the α2,6-linked SA receptor but is incompatible with the orientation of the hydrophilic glycosidic oxygen of the α2,3-linked SA receptor. The residue S228 forms a hydrogen bond with SA1, which increases the binding affinity of HA for the human receptor. Moreover, the avian H2 HA binds more efficiently to the human receptor than the avian H3 HA owing to hydrogen-bond interactions between N186 in H2 HA and Gal2, which are absent in H3 HA owing to the short side chain of S186. The structural figure was created using PDB accession 2WR7.

Figure 4: Haemagglutinin proteins from the H5 and H7 subtypes in complex with the avian and human receptor analogues.

Crystal structures of H5 and H7 haemagglutinin (HA) proteins in complex with avian and human receptor analogues. a | Wild-type H5 HA contains the residue Q226 and is glycosylated at residue N158. It has the capacity to bind preferentially to the avian receptor (α2,3-linked sialic acid (SA) receptor; shown in cyan) in a favourable trans conformation, but it binds weakly to the human receptor (α2,6-linked SA receptor; shown in magenta) in an unfavourable trans conformation. The hydrophilic residue Q226 provides a hydrophilic environment, which is compatible with the hydrophilic glycosidic oxygen atom of the avian receptor and incompatible with the hydrophobic C6 atom of the human receptor. Thus, the H5 HA preferentially binds the avian receptor. The structural figures were created using the Protein Data Bank (PDB) accessions 4K63 and 4K64. b | The ferret-transmissible mutant H5 HA (H5mut) has the residue L226 and residue 158 is deglycosylated. It binds preferentially to human receptor in a favourable cis conformation, whereas it binds weakly to the avian receptor in an unfavourable cis conformation. The hydrophobic residue L226 creates a hydrophobic environment, which is compatible with the hydrophobic C6 atom and incompatible with the hydrophilic glycosidic oxygen atom. Thus, H5mut preferentially binds to the human receptor. The structural figures were created using PDB accessions 4K66 and 4K67. c,d | The crystal structures of Anhui-H7N9 HA (AHH7, carrying the four amino acid substitutions S138A, G186V, T221P and Q226L) and an Anhui-H7N9 HA mutant (AHH7mut), in which L226 has been mutated to Q226, in complex with the avian or human receptor analogues provide insights into the structural basis of the shift in receptor binding. AHH7 and AHH7mut bind to both avian and human receptor analogues. The avian receptor analogues bind in different conformations to AHH7 (cis) and the AHH7mut carrying Q226 (trans), whereas the human receptor analogue binds to both proteins in a cis conformation. The four amino acid substitutions in AHH7 create a more hydrophobic environment that is favourable for binding to the human receptor. Furthermore, mutagenesis assays showed that the amino acid substitution Q226L is not solely responsible for the shift in receptor-binding preference of AHH7, and other substitutions also have a role. The structural figures were created using the PDB accessions 4KOM, 4KON, 4LKJ and 4LKK.

Summary and conclusions

The determinants that contribute to the host jump of avian influenza A viruses are complex and involve several viral and host factors. Recent crystallographic studies have provided molecular insights into the shift in receptor binding caused by mutations in the viral envelope protein HA, which is a major (but not the only) determinant of the host switch. To efficiently switch, viruses must acquire a preference for the human receptor, or at least the ability to bind weakly to the human receptor in addition to the avian receptor to successfully infect and replicate in human epithelial cells in the URT, which predominantly express the α2,6-linked SA receptor. It is likely that decreased binding to avian receptors is required for human-to-human transmission. This is because the human URT is covered with secreted mucin molecules that contain the α2,3-linked SA receptor, so viruses could get trapped in the URT as these mucin molecules (containing attached virus) are tightly bound to the respiratory epithelium and are unlikely to be transmitted in droplets generated by coughing or sneezing⁹¹.

 

Amino acid substitutions in HA have been identified as major determinants for preferential targeting of human, rather than avian, receptors. The H1, H2 and H3 subtypes of influenza A viruses have naturally adapted to humans, causing worldwide pandemics and epidemics. In the H1 subtype, the amino acids at positions 190 and 225 in the receptor-binding sites seem to be important for the shift in receptor-binding specificity and, in addition, different combinations of mutations result in altered receptor-binding specificity: H1 HA proteins that contain E190/G225, E190/D225 or D190/G225 in their receptor-binding site have dual receptor-binding specificity, whereas those that contain the D190/D225 and D190/E225 substitutions specifically bind to the human receptor. The Q226L and G228S substitutions in the HA glycoproteins of H2 and H3 subtypes are sufficient to change the receptor-binding preference from the avian receptor to the human receptor. Moreover, experimental adaptation of the H5 subtype showed that the Q226L substitution and loss of a glycosylation site near the receptor-binding site contribute to the shift in receptor-binding preference from avian to human. Finally, in the H7 subtype, amino acid substitutions at positions 186 and 226 increase binding to the human receptor; however, H7 HA still preferentially binds to the avian receptor, and the amino acid substitutions that are responsible for the shift in receptor-binding specificity remain to be determined.

 

Although the molecular bases of the receptor-binding preference shifts for H1, H2, H3 and H5 HA proteins have been established, the mechanism for other HA subtypes remains unknown, particularly for H7 and H9. In the future, more efforts are needed to elucidate the molecular basis of the host jump of the other HA subtypes, which should aid the rapid identification of newly emerging epidemic and pandemic strains. Furthermore, owing to technical limitations, we can currently only solve the structure of HA in complex with simple receptor analogues with pentasaccharide or trisaccharide. However, the sialylated glycan receptors in different hosts and tissues are far more complex, with different oligosaccharide chains in the receptor, and the molecular interaction between HA and complex sialylated glycan receptors should be studied in the future.

 

In conclusion, as single amino acid changes seem to be sufficient to alter receptor-binding preference, and as natural selection is unpredictable, extensive surveillance of influenza viruses will be crucial for the prevention and control of future pandemics.

 

Yi Shi,^{1, 2,} Ying Wu,^2, Wei Zhang,^2, Jianxun Qi^2, & George F. Gao^{1, 2, 3,} 

                                                                                                                                                                                                                                                                                                                                                                                                                                 

Nature Reviews Microbiology | Review

Nature Reviews Microbiology (20140 Volume: 12, Pages: 822–831

DOI: doi:10.1038/nrmicro3362

Published online 10 November 2014

 

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The emerging role of extracellular vesicles as biomarkers for urogenital cancers

Extracellular vesicles mediate intercellular signaling and are potential sources of cancer biomarkers. Nawaz and colleagues describe the biogenesis of extracellular vesicles, and the methods available for their isolation and characterization. The authors also discuss current research into the identification of vesicle-derived biomarkers for cancers of the prostate, kidney and bladder.

Abstract

The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signaling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.

Key points

• Extracellular vesicles are small (40–5,000 nm diameter) membrane-bound vesicles that can be categorized into exosomes, microvesicles and apoptotic bodies according to their size, origin, morphology and mode of release
• Whereas the generation of exosomes involves endocytosis, formation of multivesicular bodies and subsequent membrane fusion, microvesicles are produced by membrane budding and apoptotic bodies result from membrane blebbing during apoptosis
• Over the past 10 years, various methodologies for the effective isolation of extracellular vesicles have been developed, including centrifugation, affinity capture, precipitation and the use of microfluidic devices
• Extracellular vesicle cargo is thought to reflect the cell-type of origin, suggesting it could be a promising source for the discovery of novel biomarkers

 

Introduction

 

Urogenital cancers—cancers of the reproductive and renal organs—are major causes of morbidity and mortality worldwide.^{1, 2} The multistage, stochastic and heterogeneous nature of these malignancies, resulting from genetic and epigenetic modifications, poses a fundamental challenge to monitoring. Although surgical treatment and chemotherapy for urogenital cancers have improved in the last decade, the prognoses for these diseases remain poor, as existing tests are not sufficiently sensitive or specific to diagnose urogenital cancers at early stages, and none has been shown to significantly decrease overall mortality. Current diagnostic procedures include general examinations and biopsies, such as image-guided prostate biopsy,³ cystoscopy and transurethral resection of the bladder,⁴ nephrectomy and percutaneous renal tumour biopsies,⁵ all of which lack sensitivity and can be associated with significant health complications (for example, biopsies are invasive procedures associated with bleeding and risk of infections). Moreover, the location of urogenital cancers deep within the pelvic region makes them hard to access. Thus, in the absence of early symptoms, cancers are diagnosed at an advanced stage, by which time patients have poor outcomes and tumours have often metastasized.

 

Extracellular vesicles have gained considerable attention in the past 10 years as potential sources for biomarker discovery. These small (40–5000 nm diameter) membrane-bound vesicles are categorized into exosomes, microvesicles or ectosomes, apoptotic bodies^{6, 7, 8, 9, 10} or Golgi vesicles¹¹ on the basis of their size, origin, morphology and mode of release. Well-known for biological effects, such as signalling and transfer of cargo, extracellular vesicles are secreted under various pathophysiologic conditions into the extracellular environment by a variety of cell types, promoting tumour progression, survival, invasion and angiogenesis,^{12, 13, 14, 15, 16, 17} as well as influencing the immune response, cell-to-cell communication, extracellular matrix degradation, coagulation, stem-cell renewal, cardiovascular functions and resistance to drugs (Figure 1).^{18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30}

 

Figure 1: Classes of extracellular vesicles.

Extracellular vesicles comprise a heterogeneous mixture of exosomes, microvesicles and apoptotic bodies. The biogenesis of these three subtypes differs: microvesicles bud directly from the plasma membrane, whereas exosomes are formed by endocytosis and the subsequent formation of multivesicular bodies, and apoptotic bodies are formed as a consequence of apoptotic disintegration. Extracellular vesicles regulate numerous biological functions, such as cell-to-cell communication and horizontal transfer of cargo, and have been implicated in a number of biological pathways.

Surprisingly, the biomolecular cargo of extracellular vesicles is stable in biological fluids and protected against exogenous RNases and proteases, owing to its encapsulation within membrane vesicles,^{23, 31, 32} or association with RNA-binding or DNA-binding proteins^{33, 34, 35} or lipoprotein complexes.^{36, 37} Thus, extracellular vesicles might be stable under adverse physical conditions, such as extremes in pH, long-term storage and multiple freeze–thaw cycles,^{33, 38} making them an appealing source for biomarker development.

 

Figure 2: Extracellular vesicle biogenesis.

Extracellular vesicles originate through different mechanisms. a | Exosomes initiate as intraluminal vesicles that are formed by endocytosis in response to pathogens, ligands or other stimuli; these endocytic vesicles mature to early endosomes, and then into late endosomes, or MVBs. Following the ubiquitin-dependent interactions with ESCRT complexes, MVBs can be sorted for lysosomal degradation or they can fuse with the plasma membrane and be released as exosomes. ALIX binds to MVB cargo, preventing lysosomal degradation and favouring exosomal release. Rab GTPases regulate MVB fusion with the plasma membrane and release of exosomes. b | Microvesicles are formed by the outward budding and fission of plasma membrane lipid microdomains, which is controlled by regulatory proteins and cytoskeleton elements, that promote membrane curvature at ceramide-enriched domains (blue bars), resulting in microvesicle budding. After synthesis in the ER, protein cargo is transported to the Golgi apparatus, modified and packaged into small vesicles secreted as transport Golgi vesicles. c | Cells undergoing apoptotic disaggregation produce large membrane blebs, known as apoptotic bodies or apoptosomes. Abbreviations: ALIX, ALG-2-interacting protein X; ER, endoplasmic reticulum; ESCRT, endosomal sorting complexes required for transport; MVB, multivesicular body; TSG101, tumour susceptibility gene 101 protein.

Several reports indicate that cancer cells release more extracellular vesicles than normal cells,^{17, 39, 40} and that the biomolecular cargo (that is, proteins, nucleic acids and lipids) is reflective of the cell of origin.^{41, 42} Consequently, knowledge about the content of extracellular vesicles derived from tumour cells with differing stages of aggression could be used to establish new diagnostic approaches using patient-derived vesicles from body fluids. The detection of biomarkers in body fluids has major advantages over the use of tissue markers, which most often require invasive biopsies that can be difficult to perform and potentially dangerous. Urine-based tests, in particular, could offer attractive approaches for large-scale screening, as large amounts of urine can be collected longitudinally. Ultimately, discriminating between cargoes associated with extracellular vesicles in body fluids using proteomic and genomic profiling approaches could provide insight into disease staging. An important first step is to develop sensitive, rapid and highly effective strategies to enable the collection of extracellular vesicles, and to adapt standardized procedures for routine clinical diagnostic application.

 

In this Review, we provide a comprehensive overview of the roles of extracellular vesicles in the most common urogenital cancers (prostate, kidney and bladder). This includes a detailed overview of the current knowledge of the different classes of extracellular vesicles, their biogenesis, potential biological functions and available technologies for isolation and downstream analyses. Existing knowledge regarding the cancer-specific biology of extracellular vesicles, and their potential use as vehicles for biomarker discovery, are reviewed and discussed.

 

Figure 3: Multistep validation of biomarkers from extracellular vesicles.

A potential flowchart for the validation and clinical implementation of biomarkers based on extracellular vesicles. The flowchart shows a step-by-step process by which the profiling and discovery of exosomal cargo molecules could ultimately be translated into a clinically applicable biomarker signature. At each step defined goals and criteria must be met in order to proceed to the next level.¹⁶³

Conclusions

Despite the considerable research efforts applied to cancer biomarker discovery, a deficit of reliable markers to facilitate early detection, accurate prognosis and reliable prediction of response to treatment still remains. The quest for clinically relevant biomarkers for urogenital cancers remains an unmet challenge. Ongoing efforts for the identification of biomarkers include a variety of profiling technologies that are aimed at the discovery of genetic/epigenetic, proteomic and lipidomic alterations. Although lipidomic analyses of extracellular vesicles are still relatively rare, we expect that the recently revived interest in cancer metabolism will result in an increase in such studies in the future.¹⁶⁰

 

The identification of molecular signatures in biological fluids could create 'liquid biopsies', which would effectively overcome many of the challenges associated with traditional tissue sampling (such as invasiveness and tumour heterogeneity). In this regard, the analysis of extracellular vesicles derived from body fluids could offer an especially attractive source of biomarkers, since these vesicles are thought to reflect the molecular composition of the secreting cell. In urogenital cancers, increased levels of extracellular vesicles during tumorigenesis might serve as indicators for disease surveillance. Interestingly, molecular cargoes, such as nucleic acids and proteins, seem to be selectively sorted into extracellular vesicles, as recently shown for neural precursor cells exposed to proinflammatory cytokines,¹⁶¹ and these regulated mechanisms in cancer cells can provide access to a tumour-specific repertoire. For instance, several proteomic studies have revealed the presence of urinary extracellular vesicles that contain candidate proteins unique to cancer types that include a broad range of urogenital diseases.^{25, 26, 29, 102, 110, 112, 147, 157, 162} Such tumour-specific vesicles are easily captured from urine using established isolation procedures, which enables repeated tissue sampling.

 

One of the current challenges for the implementation of biomarkers based on extracellular vesicles in clinical practice is the development of isolation and detection methods that are compatible with current practices (Figure 3). The development of robust techniques and sensitive capture platforms that use readily accessible body fluids, particularly urine, could offer novel approaches for disease staging and diagnosis. Current efforts to systematically catalogue the nucleic acid, protein and lipid constituents of extracellular vesicles isolated from richly annotated clinical samples could ultimately help in developing sensitive and selective capture platforms directed towards specific extracellular vesicle subpopulations. Advances in next-generation sequencing and mass-spectrometry-based proteomics and metabolomics are likely to enable appropriate candidates to be established in the near future.

 

Muhammad Nawaz,^1, Giovanni Camussi,^2, Hadi Valadi,^3, Irina Nazarenko,^4, Kari Xiaoqin Wang,^3, n Ekström,^3, Simona Principe, Neelam Shah,^6, Naeem M. Ashraf,^7, Farah Fatima,^1, Luciano Neder^1, & Thomas Kislinger^5,                  

 

Nature Reviews Urology | Review

Nature Reviews Urology (2014) DOI:doi:10.1038/nrurol.2014.301                

Published online 18 November 2014

                                                                                                                                                                                                                                                                                                      

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