General Pathophysiology of Astroglia

Abstract

Astroglial cells are involved in most if not in all pathologies of the brain. These cells can change the morpho-functional properties in response to pathology or innate changes of these cells can lead to pathologies. Overall pathological changes in astroglia are complex and diverse and often vary with different disease stages. We classify astrogliopathologies into reactive astrogliosis, astrodegeneration with astroglial atrophy and loss of function, and pathological remodelling of astrocytes. Such changes can occur in neurological, neurodevelopmental, metabolic and psychiatric disorders as well as in infection and toxic insults. Mutation in astrocyte-specific genes leads to specific pathologies, such as Alexander disease, which is a leukodys-trophy. We discuss changes in astroglia in the pathological context and identify some molecular entities underlying pathology. These entities within astroglia may repent targets for novel therapeutic intervention in the management of brain pathologies.

7.1. Prologue: Neuroglia in Neurological Diseases

The role of neuroglia in neurological disorders have been widely accepted by leading neuroanatomists and neurologists of the nineteenth century, from Rudolf Virchow (who indicated that the ‘interstitial tissue of the brain and spinal marrow are one of the most frequent seats of morbid change’ [222]) to Carl Fromann, Alois Alzheimer and Nicolas Achucarro [1, 6, 8, 77], to name but a few. The neuropathological philosophy of the twentieth century was dominated by neurono-centric views, while the last decade witnessed the resurgence of neurogliopathology. Recently, the pathological potential of neuroglia in general, and astrocytes in particular, has been extensively studied and the fundamental principles of astrogliopathology have been defined [33, 74, 79, 152, 156, 173, 191, 192, 216, 219, 220, 234].

Neuroglial cells are primary homeostatic and defensive cells of the nervous system; and naturally, all types of glia are contributing to neuropathological developments. Astrocytes are a part of neural networks; they interact with neurones, with other glia and with blood vessels, thus, maintaining the structural and functional integrity of the neural tissue. Astrocytes are indispensable for maintaining neuronal functional and neuronal survival both in physiology and in pathology [214]. Therefore, astroglial failure creates a disease-permissive landscape and underlies neuronal malfunction, neuronal death and neurological deficits.

7.2. Principles of Astrogliopathology

Pathological changes in astroglia in neurological diseases are complex and diverse (Fig. 7.1). These changes can be generic or disease-specific. They often vary at different disease stages. In the context of human pathology, changes are affected by age and comorbidity. Astrogliopathology is classified into (i) reactive astrogliosis; (ii) astrodegeneration with astroglial atrophy and loss of function; and (iii) pathological remodelling of astrocytes (Fig. 7.1, [74, 156, 220]); all these pathological reactions occur together or in isolation.

Fig. 7.1.

Principles of astrogliopathology. Astrocytes undergo several types of morpho-functional changes in the brain pathology (see text for details). AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; FTD, fronto-temporal dementia

7.2.1. Reactive Astrogliosis

Reactive astrogliosis is observed in many neurological disorders. Until very recently, astroglial reactivity was considered the sole manifestation of astrogliopathology. From histopathological point of view, astroglial reactivity is characterised by mor phological hypertrophy and up-regulation of two major cytoskeletal intermediate filaments/proteins, glial fibrillary acidic protein (GFAP) and vimentin [95, 155, 191]. Reactive astrocytes undergo a variety of substantial modifications, showing multiple phenotypes with both neuroprotective and neurotoxic features. These phenotypes arguably are disease-specific, although they all can share some common properties [120, 121, 156]. Transcriptomes of reactive astrocytes in ischemia and endotoxin activation, for example, show significant differences [233].

Conceptually, reactive astrogliosis represents an evolutionary conserved (the first manifestations of astroglial reactivity are observed in many invertebrates including annelids and insects) defensive reprogramming of astroglia aimed at: (i) increased neuroprotection and trophic support of the nervous tissue; (ii) isolation of the lesioned area; (iii) reconstruction of the compromised blood–brain barrier; and (iv) facilitating the post-lesion regeneration of the nervous tissue [7, 156, 191]. The astrogliotic programme, therefore, has a high degree of flexibility and tailors functional and biochemical reprogramming of astrocytes to the nature and strength of the insult. Even within the same lesioned regions, astrocytes demonstrate a degree of heterogeneity in expression of transcription factors, inflammatory agents and signalling molecules [78, 92].

The initiation of astrogliosis is regulated mainly by damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). The DAMPs are endogenous molecules released from damaged or dying cells (ATP being the most prominent example), blood-borne factors that infiltrate brain parenchyma, etc. The PAMPs are exogenous molecules associated with infectious invaders such as bacteria or viruses; they mostly act through Toll-like receptors (TLRs) widely expressed in astrocytes [99, 203]. Astroglial cells express a wide range of receptors to both DAMPs and PAMPs: P2X₇ purinoceptors, TLRs, nucleotide-binding oligomerisation domains (NOD)-like receptors (NLRs), double-stranded RNA-dependent protein kinase, scavenger receptors, mannose receptor and receptors for complement components and mediators, such as CXCL10, CCL2, interleukin-6 and B-cell-activating factor of the tumour necrosis factor (TNF) family [70]. Often, exposure of astrocytes to DAMPs and PAMPs evokes cytosolic Ca²⁺ increases due to its release from the endoplasmic reticulum (ER) intracellular store. These Ca²⁺ signals are critical for instigating the astrogliotic programme. For instance, genetic deletion of predominant astroglial Ca²⁺ release channel of the ER, inositol 1,4,5-triphospate receptor type 2, suppresses astrogliotic response [101]. Similarly, pharmacological inhibition of Ca²⁺ release from the ER restrains astroglial reactivity triggered by amyloid-β [2]. Stimulation of astrocytes with ATP (a classical DAMP) not only triggers Ca²⁺ signalling [34, 211] but also induces formation of inflammasomes comprised of the NLR protein-1 or −2 LR, the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) and caspase-1. Activation of these inflammasomes leads to the processing of inflammatory caspase-1 and interleukin-1β (IL-1β) [137].

Reactive astrogliosis is classified according to the morphological properties and severity (Fig. 7.1). From the morphological perspective, astrogliosis is divided into isomorphic and anisomorphic astrogliosis. The isomorphic astrogliosis preserves astroglial territorial domains and it is fully reversible, whereas anisomorphic astrogliosis proceeds with violation of territorial domains, cell migration and territorial overlap, formation of astroglial palisades and ultimately the scar formation [156]. According to the severity, astrogliosis is classified into (i) mild to moderate astrogliosis; (ii) severe diffuse astrogliosis; and (iii) severe astrogliosis with compact scar formation [190, 191]. Fundamentally, astrogliosis provides for defence of the nervous tissue; it increases neuroprotection and is ultimately important for post-lesion regeneration. Even scar formation carries clear definitive function isolating the damaged area of the CNS and saving the whole at the expense of its part [156, 210]. Suppression of reactive astrogliosis usually exacerbates the course of pathology [156]. Inhibition of astroglial reactivity enlarges the size of the traumatic lesions and aggravates neurological deficit [148]. Deletion of GFAP and vimentin, both being critical for the execution of astrogliotic programme, facilitates the development of ischaemic infarcts [116] and exacerbates post-traumatic synaptic loss [154]. Ablation of astroglial reactivity increased the accumulation of β-amyloid and reduced microglial association with senile plaques in the animal model of the Alzheimer’s disease [157]. Nonetheless, in conditions of prolonged stress or severe damage, reactive astrocytes may acquire neurotoxic potential and astrogliosis as a process can become maladaptive [155].

7.2.2. Astroglial Atrophy

Astrodegeneration is a widespread class of astrocytopathy, which is represented by morphological atrophy, increased astroglial death and hence decrease in astroglial density and an impairment of homeostatic functions. Astrodegeneration has been observed in various types of neuropathologies [90, 212, 221]. Astrodegeneration is particularly prominent in major psychiatric diseases. For instance, schizophrenia, major depressive disorder, Wernicke–Korsakoff encephalopathy, and addictive disorders are all accompanied with a reduction in the packing density of astrocytes and a failure of their homeostatic cascades, the latter most notably associated with glutamate homeostasis and glutamate–glutamine shuttle, which are both impaired [50, 53, 54, 134, 163, 165, 178, 218]. Aberrant astroglial glutamate transport and catabolism are arguably responsible for abnormal neurotransmission as well as for excitotoxic neuronal death, both resulting in psychotic symptoms. In amyotrophic lateral sclerosis, insufficient astroglial glutamate clearance from the extracellular space instigates excitotoxic death of large motor neurones [177, 207], whereas in Alzheimer’s disease, reduced astroglial synaptic coverage contributes to early synaptic extinction and cognitive deficiency [215].

7.2.3. Pathological Remodelling of Astrocytes

Pathological remodelling of astrocytes covers abnormalities associated with an acquisition of abnormal molecular cascades or functional properties, which drive pathology [74, 156]. Pathological remodelling of astroglia contributes to various leukodys-trophies, most notably to Alexander disease, megalencephalic leukoencephalopathy with subcortical cysts or vanishing white matter syndrome, in all of which the astrocytopathy initiates lesions of the white matter [113]. In Alexander disease, astroglial expression of sporadically mutated GFAP gene results in early and severe leukomalacia [131]. Pathological remodelling in astroglia has been also described in mesial temporal lobe epilepsy, in which astrocytes acquire aberrant morphology, reduce gap junctional coupling and down-regulate expression of K_ir4.1 channels; all these changes compromise K⁺ homeostasis thus contributing to the initiation of seizures [19].

7.3. Astrogliopathology in Neurological Diseases

7.3.1. Neurotrauma

Traumatic injury of the brain and of the spinal cord are classified according to their nature (penetrating wounds or concussions; the later when occurring in the cervical spinal cord is known medically as cervical cord neurapraxia), their severity (mild, moderate or severe), volume (focal or diffuse), outcome (death, vegetative state, severe disability, moderate disability and good recovery) and anatomical localisation. According to its very nature, a traumatic injury to the CNS has a complex pathophysiology associated not only with direct damage to neural cells, but also with a damage to the whole organ with destruction of the blood–brain barrier and blood vessels, ischaemic insults, opening the way for secondary infection, etc. Neurotrauma predominantly triggers astrogliotic response; reactive phenotypes, however, very much depend on the pathological context [32, 33] with the severity of the damage and its anatomical localisation affecting astroglial activation.

In the healthy brain, astrocytes form numerous barriers with blood vessels and with cerebrospinal fluid; endfeet of astroglial cells together with the parenchymal basement membrane create glia limitans, which physically separates the brain parenchyma from blood vessels, perivascular spaces and the meninges. In response to a neurotrauma, an astrogliotic scar barrier is formed that delineates and isolates the areas of a focal damage from the healthy brain. Suppression of astrogliosis with consequent malformation of an astroglial scar markedly exacerbates tissue damage and neurological deficit [189]. The heterogeneity of reactive astroglial phenotypes very much depends on the distance to the lesion core. Close to the lesion astrocytes lose their domains, their processes overlap and the astroglial palisades are formed, reflecting anisomorphic astrogliosis. Astrocytes gather around the damaged sites and form the scar [32]. Astrocytes distant to the lesion core undergo isomorphic gliosis; they become hypertrophic and arguably neuroprotective. Contribution of astrocytes to tissue pathology in neurotrauma is multifaceted. Besides forming a protective scar astrocytes regulate inflammatory response, provide for homeostatic protection of the nervous tissue through the removal of extracellular glutamate, buffering K⁺ or releasing scavengers of reactive oxygen species and regulate post-traumatic remodelling of synaptic networks. Reactive astrocytes are indispensable in remodelling as suppression of astrogliosis down-regulates post-traumatic regeneration of synaptic connectivity and neuronal networks [7].

7.4. Infectious Diseases

7.4.1. Infection of Nervous Tissue

Infections of the CNS caused by bacteria, viruses, fungi and parasites are classified into meningitis, encephalitis or brain abscess. Not every infectious agent can invade the CNS. Rather, only certain neurotropic viruses, bacteria, fungi and parasites can penetrate into the brain and the spinal cord with relative ease. Furthermore, most of the pathogens are effectively stopped by the brain barriers [110]. Infectious agents may cross the blood–brain barrier using the paracellular route, via transcytotic mechanism, inside entering monocytes (the Trojan horse hypothesis) as well as by other mechanisms such as, for example, hijacking of β-adrenergic receptors as shown for Neisseria meningitidis (meningococcus) [47].

Neuroglial contribution to the infectious lesions of the CNS is of fundamental importance. Neuroprotective activation of astrocytes and microglia to a large extent defines the spread of infection through the nervous tissue and hence determines the outcome of the disease. The glial response, in turn, depends on the nature of an infectious agent. For example, contact of astrocytes with the Gram-positive bacteria such as Pneumococcus or Staphylococcus triggers rapid astrogliotic activation with marked cellular hypertrophy and up-regulation of GFAP expression [96] accompanied with synthesis and secretion of pro-inflammatory agents such as TNF-α, ILs and macrophage inflammatory protein 1α [122]. Activation of astrocytes by infectious agents (see for example [70, 197, 228]) is mediated mainly through pattern recognition receptors (PRRs), which are represented by TLR 2, 3, 4, 9 [65], NLRs, retinoic acid-inducible gene (RIG)-like receptors (RLRs) and cytokine receptors [108]. The NOD2 receptor, operational in astrocytes, recognises a minimal motif present in all bacterial peptidoglycans and it is required for astroglial reactive reprogramming in response to N. meningitidis and Borrelia burgdorferi [43].

Activation of astrocytes is also linked to TLR receptors [67]. Distinct TLR subtypes recognise and respond to different PAMPs. Lipopolysaccharide (LPS), for example, signals through TLR4; TLR3 is activated by double-stranded RNA; peptidoglycans interacts with TLR2, while TLR9 senses CpG DNA [40]. Activated TLRs interact with adaptor proteins myeloid differentiation factor 88 (MyD88) and/or a TIR-containing adaptor molecule, Toll/interferon-1 receptor domain-containing adaptor inducing interferon-γ (TRIF), which acts as a part of relevant signalling cascade [40]. Bacterial infection of the nervous tissue down-regulates expression of connexins hence decreasing gap junctional connectivity of astroglial syncytia [66]. Direct interaction of several bacteria such as Streptococcus pneumonia, B. burgdorferi and N. meningitides triggers astroglial reactivity as well as increases the production of pro-inflammatory cytokines and chemokines such as IL-6, TNF-α, IL-8, CXCL-1 and CXCL-10 [240]. Besides activation, astrocytes may undergo pathological remodelling and act as a reservoir for infection. Furthermore, astrocytes can promote apoptotic death of their uninfected neighbours through gap junction route [68, 240]. Astroglial reactivity, that includes overexpression of GFAP, keeps infectious process at bay. Indeed, genetic deletion of GFAP associated with suppressed astrogliosis, significantly exacerbated the neurological damage induced by intra-brain injection of S. aureus [197].

Astrocytes are fundamental players not only in bacterial but also in viral infections of the CNS. First and foremost, astrocytes can be directly infected by a virus. For example, astrocytes accumulate human immunodeficiency virus-1 (HIV-1) in a cluster of differentiation 81 (CD81)-lined vesicles. Inside these vesicles, the virus is protected from degradation [83]. The very same vesicles contributed to the secondary trans-infection of T-cells [83]. In the dementia caused by HIV brain infection, astrocytes undergo both reactive remodelling and astroglial degeneration and astroglial death. These processes may reduce homeostatic support and hence contribute to cognitive deficit [46]. The astroglial infection with HIV-1 (similarly to bacterial infection) decreased expression of connexins and syncytial connectivity [150]. In a similar manner, astrogliotic response is mounted in response to infection with the herpes simplex virus 1 (HSV1). Here, activation of astrocytes is mediated by TLR3 and it is neuroprotective. Deletion of TLR3 suppressed astrogliosis and exacerbated HSV pathology in mice [168] as well as in humans [91]. Infection with cytomegalovirus (CMV) was associated with astroglial homeostatic failure. The CMV infected astrocytes showed decreased released of thrombospondins and deficient glutamate uptake, possibly linked to an increased excitotoxicity [235, 236]. Neurotropic viruses of the family of Flaviviridaem represented by Zika virus and tick-borne encephalitis virus (TBEV) invade astrocytes by endocytosis [159, 240]. Astroglial infection with TBEV does not visibly affect their survival or function, and it is generally believed that astroglial cells act as a reservoir for this type of virus [240]. Astrocytes also represent the cellular target for some protozoan parasite, most notably for Toxoplasma gondii. Astrocytes infected with T. gondii undergo biochemical remodelling associated with up-regulated synthesis of kynurenic acid that in turn may be linked to some forms of schizophrenia, which will be discussed in appropriate section below. In addition, infection of astrocytes with this protozoan results in the loss of gap junctions [37].

7.4.2. Systemic Infections and the Brain: Sepsis-Associated Encephalopathy

Systemic inflammation frequently accompanies various infectious and non-infectious diseases including degenerative and metabolic disorders. This systemic inflammation often is manifested in the form of sepsis. Sepsis (and in particular abdominal sepsis) is frequently accompanied with an acute brain dysfunction, generally defined as sepsis-associated encephalopathy or SAE [187]. From the clinical perspective, the SAE is regarded as a sign of the severity of a septic state, which potentially worsens the prognosis [158]. The SAE is defined as a clinical syndrome associated with the general brain dysfunction that develops in sepsis in the absence of primary infection of the nervous tissue. The histopathological signs of the SAE include infarctions, petechial and small focal haemorrhages, septicembolic abscesses and septicopyemic microabscesses, disseminated intravascular coagulation (DIC) syndrome with fibrinous microthrombi, multifocal necrotising leukoencephalopathy, necrotic or apoptotic neuronal death, perivascular and cytotoxic oedema, damage of the blood–brain barrier and reactive neuroinflammation [89, 186, 187]. Sepsis is often associated with the formation of abscesses and microabscesses in the brain parenchyma, which can be regarded as directly associated with the SAE. The SAE, especially at the early stages is often associated with ‘sickness behaviour’, the syndrome accompanying system inflammation. The symptomatology of sickness behaviour syndrome includes anxiety, anorexia, anhedonia, depression, cognitive changes, including decreased concentration, learning and memory [56].

At the neurochemical level, the leading pathological changes in an SAE are represented by aberrant neurotransmission, which is responsible for cognitive and psychotic symptoms. Substantial changes in expression of main neurotransmitter receptors including receptors for γ-aminobutyric acid (GABA), serotonin, dopamine and noradrenaline have been observed in the brain in systemic infections [100, 208]. Changes in neurotransmitter homeostasis in sepsis are arguably related to alterations of amino acids levels in the blood characterised by a decrease in branched chain amino acids together with relative increase in aromatic amino acids [15]. In addition, compromised brain barriers allow a substantial influx of aromatic amino acids, such as tyrosine, phenylalanine and tryptophan, which may act as false neurotransmitters and alter biosynthesis of true neurotransmitters (e.g., dopamine, noradrenaline and serotonin—[188]).

Astrocytes, endfeet of which form glia limitans and hence can be considered as the parenchymal portion of brain barriers (the blood–brain and the blood–cerebrospinal fluid), define, to a very large extent, the resistance of the nervous tissue to the systemic inflammation. Intimate contacts of astrocytes with all other cellular elements of the nervous tissue allow them to regulate the relationship between the CNS and systemic physiology and pathology. In the context of SAE, astroglial reactivity is the principal mechanism that limits the propagation of pathological agents through the nervous tissue [41, 193]. Inhibition of astrogliotic response compromises astroglial barrier function and aggravates encephalopathy in the context of systemic inflammation or infectious lesion to the brain. For example, in transgenic mice with deleted gene for GFAP (this intervention suppresses astrogliosis), brain abscesses caused by Staphylococcus aureus or Toxoplasma gondii were much larger. Lesions become poorly demarcated, bacterial penetration significantly increased, neuronal death was much exacerbated and severe brain oedema developed [197]. Suppression of astroglial reactivity by activation of NF-κB signalling cascade in retinal ischemia or in spinal cord injury, is associated with an increased neuronal damage [27, 63]. Finally, inability of astrocytes to acquire reactive phenotype results in swelling, cytotoxic oedema and spread of damage in infectious abscesses [187].

Astrocytes contribute to the pathology of the blood–brain barrier, which is classified as disruptive and non-disruptive alterations, with both variants present in systemic inflammation [187]. The non-disruptive BBB pathology develops at the molecular and cellular levels when BBB permeability is affected following up- or down-regulation of receptors and transporters expressed in endothelial cells and astrocytes [209]. Disruptive BBB alterations develop through anatomical changes, which include degradation of glycocalyx, a loss of integrity of tight junctions, mitochondrial damage, appearance of fenestrae between endothelial cells, endothelial cells death, collapse of glia limitans and astrocytopathy. Disruption of BBB in systemic inflammation is mediated by blood-derived metalloproteinases, prostanoids, nitric oxide and reactive oxygen species [38, 136]. The switch between non-disruptive and disruptive BBB pathology depends on the severity of systemic inflammation. At the early stages of SAE the non-disruptive changes prevail, whereas in severe sepsis, both non-disruptive and disruptive changes occur [209].

7.5. Toxic Damage of the CNS

7.5.1. Heavy Metal Toxic Encephalopathies

Heavy metals, which cause severe brain damage with cognitive deficits, target primarily astrocytes. This is because heavy metals (such as manganese, lead, aluminium or mercury, in the form of methylmercury) mainly accumulate into astrocytes through different plasmalemmal transporters. In general, heavy metals down-regulate astroglial expression of glutamate transporters which decrease glutamate clearance and trigger excitotoxicity [198, 199, 217, 232].

Poisoning by methylmercury is known as Minamata disease named after the city of Minamata in Japan where the disease was first described [129]. The symptoms of Minamata disease include visual abnormalities, sensory lesions, cerebellar ataxia, hearing loss, weakness, tremor and cognitive decline. Methylmercury primarily accumulates in astroglia, where it inhibits glutamate and cystine uptake [5]. Suppression of glutamate uptake instigates exocytotic neuronal death, whereas inhibition of cystine transport limits astroglial synthesis of glutathione hence reducing astroglial capacity to counteract the accumulation of reactive oxygen species; both these processes contribute to neurotoxicity and neuronal death [57, 232].

Exposure to toxic concentrations of lead similarly causes neurodegeneration. Lead primarily accumulates in astroglia, where it down-regulates expression of EAAT-2 glutamate transporter, increases astroglial production of vascular endothelial growth factor, and impairs astroglia-associated water homeostasis by increasing the water permeability of aquaporin 4 [85]. Arguably, these mechanisms contribute to cytotoxic and vascular brain oedema observed in patients with lead poisoning.

Aluminium toxic encephalopathy is manifested by cognitive impairments, speech alterations, seizures and flapping wrist tremor (asterixis). Treatment of cultured astrocytes with aluminium led to swelling, destruction of the cytoskeleton, reduction in gap junctional connectivity, inhibition of glutamate uptake and increased astroglial apoptosis. Loss of astroglial glutamate uptake triggered neuronal death in neuronal–glial co-cultures [198, 199].

The main symptom of acute manganese neurotoxicity is an acute psychosis, whereas chronic manganese poisoning leads to parkinsonism. Astrocytes possess the high capacity manganese transport system; treatment of primary cultured astrocytes with manganese suppresses glutamate uptake and promotes apoptosis [57].

7.5.2. Hyperammonemia and Hepatic Encephalopathy

Increase in blood ammonium accompanies several diseases. The most frequent cause of hyperammonemia is, however, associated with an acute or chronic liver failure (the liver being the main organ for ammonia clearance). Hyperammonemia affects the brain and triggers a condition generally known as hepatic encephalopathy, manifested by cognitive and behavioural impairment; symptoms include confusion, forgetfulness, irritability and alterations of consciousness, such as lethargy and somnolence. Severe hyperammonemia provokes brain oedema, coma and death [31, 35, 73]. In the CNS ammonia is detoxified by glutamine synthetase localised exclusively in astrocytes; this enzyme catabolises ammonium reaction with glutamate and produces glutamine [3, 143, 175]. This reaction is central for glutamate-glutamine shuttle; it also fixes ammonium, which is liberated during physiological neuronal activity [126]. Ammonium overload occludes this pathway and blocks glutamine synthetase hence causing major disturbances of glutamatergic and GABAergic (as glutamate is the precursor to GABA) neurotransmission, which underlie all the symptoms outlined above [31, 35].

Hyperammonemia also affects homeostatic astroglial functions. Exposure of astrocytes to ammonium results in a down-regulation of expression of inward rectifying K_ir4.1 channels, an event mediated through astrocytic NMDA receptors by a yet uncharacterised mechanism. Decrease in the density of K_ir4.1 channels, in turn, affects astroglial K⁺ buffering which may impair neuronal excitability [144, 166]. Exposure of astrocytes to ammonium also produces aberrant Ca²⁺ signalling by increasing expression of Ca²⁺-permeable TRPC1 channels, up-regulating expression of Ca_v1.2 voltage-gated Ca²⁺ channels and facilitating Ca²⁺ release from the intercellular stores [86, 119, 223]. Increased Ca²⁺ load of astroglial cytosol, in turn, triggers the exocytotic secretion of glutamate which further exacerbates excitotoxic damage of the nervous tissue [82, 139]. Finally, increased ammonium compromises astroglial transport of Na⁺ and H⁺ which contributes to aberrant pH regulation in the CNS [105, 106]. All these molecular changes result in impaired synaptic transmission, synaptic plasticity and cognitive capabilities [45].

7.6. Astrogliopathology in Stroke

A disruption of the blood flow results either from a blood vessel rupture (that causes a haemorrhage), or by a restriction of blood supply to the brain or parts of the brain, because of a vascular occlusion (thrombosis or embolism), or to a systemic decrease in blood supply (resulting, for example, from a heart failure). This status is generally referred to as brain ischaemia. As a consequence, brain ischaemia can be either global, or focal, the latter corresponding to a stroke.

Astrogliopathology in stroke is complex and multifaceted, with astrocytes being both neuroprotective and neurotoxic [81, 239]. Focal ischaemia results in the infarction of nervous tissue creating a zone of pan-necrosis or an infarction core. At this core, all cells, neurones, glia and other non-neuronal cells undergo rapid necrosis. The size of the core is determined by anatomical location and duration of the ischemic attack. Quite frequently the focal ischemia is transient, as the blood flow can be restored when the vessel blockage is removed. In this case, restored blood flow results in reperfusion of the damaged area, which itself is potentially damaging because of the production of reactive oxygen species and secondary ion imbalances.The infarction core is surrounded by the ischemic penumbra, which contains viable cells, although with compromised metabolism and function. The infarction core is formed rapidly, within minutes to hours after initiation of the stroke. This is followed by a much slower process of expansion of the infarction zone through the penumbra, which develops over many hours and days. The final neurological deficit is often defined by the limits of the infarction expansion, which in turn depends on astroglial response.

Astrocytes support neurones in the ischaemic penumbra through several homeostatic pathways. First and foremost, astrocytes maintain homeostasis of glutamate in the ischaemic zones. They also feed neurones with metabolic substrates such as lactate. Energising astroglial mitochondria, for example, increase neuroprotection in the ischaemic context [180]. Taming glutamate excitotoxicity, which always follows stroke, almost solely falls onto astroglial cells. Down-regulation of expression of the astroglial glutamate transporter GLT-1/EAAT1 with siRNA increases the size of the infarct [167], whereas targeted overexpression of GLT-1 in astrocytes limits the infarction volume and alleviates neurological deficit [88]. Similarly, stimulation of glutamate uptake with pharmacological agents such as tamoxifen or riluzole decreased infarction volume in animal models [227, 238]. Of note, astroglial glutamate transporters are Na⁺ dependent, and hence maintenance of Na⁺ transmembrane gradients is critical for glutamate clearance [109]. Another important component of astroglia-dependent neuroprotection in the ischaemic penumbra is associated with antioxidant defence. Astrocytes are critical for both glutathione and ascorbic acid systems, which are the most powerful scavengers of reactive oxygen species [61, 62, 125]. Progression of cell death through the ischaemic penumbra is mediated by spreading depolarisation, which stresses astroglial ionostatic capacity. Furthermore, astrocytes may propagate death signal, triggering distant neuronal death [115, 142].

An important component of astroglial response to stroke is associated with reactive astrogliosis. Ischaemic damage to the brain tissue rapidly instigates astroglial activation through the release of DAMPs; the severity of astrogliotic remodelling and reactive phenotypes depend on the distance to the ischemic core [33]. Astrocytes close to the ischaemic core undergo anisomorphic gliosis, form astroglial palisades and produce astroglial scar that limits the damage to the nervous tissue. In parallel, distantly to the core astrocytes undergo isomorphic, neuroprotective astrogliotic remodelling, which is critically important for post-lesion regeneration The main outcome of astrogliosis in the immediate vicinity of the necrotic area is the formation of an astroglial scar, whereas more peripheral reactive astrocytes are important for post-lesion regeneration [81].

7.7. Metabolic Disorders

7.7.1. Congenital Glutamine Deficiency with Glutamine Synthetase Mutations

Congenital glutamine synthetase deficiency, a rather rare recessive inborn disease, results from mutations to the gene GLUL that encodes astroglia-specific glutamine synthetase, thus, this disorder can be considered as a specific astrogliopathy. This disease is characterised by pronounced malformation of the brain with severe white matter deficiency and abnormal gyri formation. Functionally, this deficiency is manifested as epileptic encephalopathy. The deficit in glutamine synthetase in the liver promotes chronic hyperammonemia. In addition, levels of glutamine are reduced in the brain as well as in other organism fluids. The disease results in prenatal malformation of various organs and is generally incompatible with life. Most of the infants die shortly after birth. The leading pathophysiological mechanism is associated with impaired ability of astrocytes to produce glutamine, which affects excitatory and inhibitory transmission; in addition, deficient glutamine synthetase cannot properly detoxify ammonium [195].

7.7.2. Pyruvate Carboxylase Deficiency

Pyruvate carboxylase is an enzyme of gluconeogenesis and it also contributes to anaplerotic metabolic pathways (i.e. producing intermediates for metabolic chains such as the Krebs cycle). In the CNS, pyruvate carboxylase is predominantly expressed in astrocytes. Pyruvate carboxylase deficiency is an autosomal recessive disease associated with impaired metabolism. The symptoms include retardation of mental development, recurrent seizures and metabolic acidosis [127]. There are three clinically distinct forms: type A, or the infantile form, in which children die in the early years; type B, or the severe neonatal form, with many neurological signs including pyramidal symptoms, in which babies die within 3 months after birth; type C or the benign form, which is characterised by mild neurological developmental deficits. The cellular pathogenesis remains largely unknown, but it is probably linked to reduced astroglial homeostatic function, such as glutamate buffering and regulation of angiogenesis [57]

7.7.3. Niemann–Pick Type C Disease

Niemann–Pick disease type C is a progressive neurodegenerative disease associated with hepatosplenomegaly. It is characterised as an autosomal recessive lysosomal storage disease, which results from loss-of-function mutations of genes encoding NPC-1 or NPC-2 proteins [176]. These proteins are localised in astroglial perisynaptic processes and may be involved in the regulation of cholesterol transport and, hence, synaptogenesis or synaptic maintenance [153]. Astroglia-specific genetic deletion of Npc1 from mice resulted in reduced neuronal cholesterol, which was associated with decreased neuronal and glial death and three times increase in the life span [237]. There is also evidence of a possible contribution for NPC-1 protein in calcium homeostasis and signalling.

7.7.4. Aceruloplasminemia

The enzyme ceruloplasmin (also known as ferroxidase) is a part of iron metabolism. In the CNS this enzyme is expressed almost exclusively in perivascular astrocytes. Ceruloplasmin is an important component of protection of the nervous tissue against iron-associated lipid peroxidation and formation of hydroxyl radicals. Mutation of the ceruloplasmin gene with loss-of-function causes the autosomal recessive disease known as aceruloplasminemia, which can be defined as an inherited neurodegenerative disorder with systemic iron-overload syndrome [138]. This disease is characterised by primary lesions to astrocytes, which affects their morphology and results in an appearance of foamy spheroid bodies at the vascular endfeet [147]. Aceruloplasminemia is also associated with neuronal death and the appearance of iron deposition.

7.8. Alexander Disease

Alexander disease (AxD), named after William Stewart Alexander, a neuropathologist who described it for the first time [4], is a rare, chronic and usually fatal neurodegenerative disorder. Clinically, AxD may be defined as a severe leukodystrophy; pathophysiologically, it is a primary genetic astrogliopathology [131]. The AxD results from a dominant gain-of-function mutation of the gene encoding GFAP. This leads to astroglial pathology that, in turn, results in a severe damage to the developing white matter. The histopathological hallmark of AxD is an accumulation of protein aggregates, known as Rosenthal fibres, around astroglial nuclei and endfeet [131]. AxD is subclassified into: (i) Type I, characterised with an early onset and severe mental and physical disabilities, megalencephaly, seizures, spasticity, difficulty speaking and swallowing, and (ii) Type II, with a later onset and somewhat different and milder clinical manifestations with normal development and head size, with rare occurrence of seizures, but with ataxia, visual and autonomic abnormalities, troubles in sleeping patterns, hyperreflexia, difficulty speaking and swallowing [160].

Astrocytes in AxD demonstrate reactive morphology. These glial cells also remodel their biochemistry and secretome. In particular, astrocytes start to release pro-inflammatory factors TNF-α and IL-1β. In addition, astrocytes in AxD have reduced expression of glutamate plasmalemmal transporters, decreased activity of proteasomes, increased autophagy and increased activity of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) pathway [131]. Multiple mechanisms by which pathological mutation of GFAP affects cellular functions have been considered. These include: (i) mutated GFAP through positive feedback loop inhibits proteasome function which activates JNK, and activated JNK directly further inhibits proteasome [204]; (ii) mutated GFAP inactivates one or more proteins by degradation of the Rosenthal fibres, where fragments of the small stress proteins, HSP27, αB crystalline, the 20S proteasome subunit, p-JNK, p62 and plectin, have been detected [131]. So far the AxD remains incurable, although several therapeutic strategies aimed at reducing GFAP expression are in development.

7.9. Neurodevelopmental Disorders

7.9.1. Autism Spectrum Disorders (ASD)

The class of autistics spectrum disorders (ASD) embraces numerous pathological conditions of heterogeneous clinical presentation and pathophysiology. They all, however, are manifested by deficits in social interactions and restrictive patterns of behaviours. Some of the autistic diseases are associated with intellectual deficits [162]. The underlying mechanism of ASDs is most likely associated with malformation of neuronal networks and aberrant neurotransmission in embryonic development caused by environmental and/or intrinsic factors [42, 80, 130, 174]. Formation of neuronal ensembles, synaptogenesis and synaptic elimination all critically depend on the performance of the astroglial cradle, which controls birth, life and death of synapses [213]. Astrocytes are responsible for neuroprotection and detoxification of harmful agents, including reactive oxygen species (through secreting antioxidants such as glutathione and ascorbic acid [30, 229]). Astrocytes tame excitotoxicity through glutamate uptake and they also control neurotransmitters catabolism and supply of neurotransmitter precursors [214]. In parallel, astrocytes are the main target for neurotoxic factors, such as heavy metals, which are linked to the aetiology of ASD [234]. Astrogliopathology in ASD has not been investigated in great details; there are some indications for astrogliosis [234], increased expression of connexin 43 and decreased expression of aquaporin 4 [71].

7.9.2. Down Syndrome

Down syndrome (DS), which is linked to the trisomy of chromosome 21, is characterised by mental retardation. In DS, the density of astrocytes is significantly reduced in the cortex [102] with decreased ability to properly support synaptogenesis and neuronal maturation [44].

7.9.3. Fragile X Syndrome

Expression of Fragile X mental retardation protein (FXMRP) causes a specific form of a neurodevelopmental disease manifested in ASD symptoms and mental disability, Fragile X syndrome that is also known as Martin–Bell syndrome or Escalante’s syndrome [107]. Expression of FXMRP in astrocytes weakens their homeostatic function and neuroprotection in the in vitro experiments. Co-culturing healthy neurones with astrocytes harbouring FXMRP leads to abnormal neuronal dendritic morphology and reduced synaptic connectivity. In contrast, co-culturing FXMRP expressing neurones with healthy astrocytes prevents the development of abnormal dendritic morphology [97, 98].

7.9.4. Costello Syndrome

Costello syndrome (named so after its discoverer Jack Costello [48]) belongs to the family of the so-called RASopathies (where RAS stands for rat sarcoma) characterised by aberrant Ras signalling [205]. In this pathology, astroglial cells expressing a mutated HRAS (Harvey rat sarcoma viral oncogene homolog) gene demonstrate hyperactive Ras signalling, which accelerates differentiation and maturation of astrocytes, and leads to astroglial hypertrophy. This is also associated with pathological extracellular matrix and abnormal formation of neuronal networks that in turn causes cognitive and behavioural abnormalities [112].

7.10. Major Neuropsychiatric Diseases

7.10.1. Schizophrenia

In schizophrenia the wide spectrum of astroglial abnormalities is present. Conceptually, schizophrenia is associated with astroglial asthenia, atrophy, loss of homeostatic capabilities and arguably pathological remodelling, while reactive changes are not characteristic. Decrease in astroglial numbers, as well as dystrophic or swollen astroglial profiles, appear in various brain regions, including cortical and hippocampal structures [69, 163, 181, 226]. Astrocytes derived from human induced pluripotent stem cells obtained from schizophrenic patients and injected into mice, demonstrated atrophic morphology and loss of homeostatic functions [230].

Astrocytes in schizophrenia are characterised by a significant down-regulation of expression of several astroglia-specific molecules such as deiodinase type II, aquaporin-4, S100β, glutamine synthetase, plasmalemmal glutamate transporters and thrombospondin. These changes were the most prominent in the deep layers of the anterior cingulate gyrus, suggesting that a subset of astrocytes localised to specific cortical layers can be affected [231]. In the prefrontal cortex and hippocampus, a decrease in the expression of EAAT1/2 plasmalemmal glutamate transporters has been detected [16, 17, 146, 185], which may be linked to abnormalities in glutamatergic transmission. Genetic deletion of EAAT1 glutamate transporter promoted appearance of schizophrenia-like phenotypes manifested by locomotor hyperactivity and abnormal social behaviour [103, 104]. Astrocytes from rodent phencyclidine model of schizophrenia demonstrated a decrease in the expression of plasmalemmal cystine–glutamate exchanger Sxc⁻ [12], which modulates extrasynaptic concentration of glutamate and contributes to the biosynthesis of glutathione. Astrocytes may promote aberrant neurotransmission through synthesis and release of kynurenic acid that acts as an endogenous inhibitor of the NMDA receptor glycine binding site; kynurenic acid also blocks acetylcholine nicotinic receptors. The astroglial production of kynurenic acid is significantly up-regulated following brain infection with Toxoplasma gondii, which increases the risk of schizophrenia [183].

7.10.2. Mood Disorders

Astrogliopathology seems to be rather prominent in mood disorders [165, 178, 218]. The total number of glial cells and of astrocytes, in particular, is decreased in the orbitofrontal area and anterior cingulate, prefrontal, entorhinal and subgenual cortices, as well as the amygdala of the brains obtained from patients with major depression or bipolar disorder. [26, 49, 50, 149, 164]. In animals subjected to chronic stress, which instigates depressive phenotypes, GFAP expression and number of GFAP positive cells were reduced [28, 55]. Similarly, in models of attention deficit disorder and depression other astroglial markers, including aquaporin 4, astroglial connexins, astroglial plasmalemmal glutamate transporters and glutamine synthetase were all down-regulated [14, 21, 184].

Ablation of astrocytes in the medial prefrontal cortex of mice with the neuroglial toxin L-α-aminoadipic acid triggered an emergence of a depressive phenotype similar to that induced by chronic stress [13]. Exposure to chronic stress led to a down-regulation of astroglial expression of connexin 43 along with the reduction of gap junctional coupling in astrocytic syncytia. Pharmacological inhibition of astroglial connexon-based channels in the prefrontal cortex induced depressive behaviour manifested by anhedonia [201]. A similar phenotype was observed after inhibition of astroglial plasmalemmal glutamate transporters [18]. Chronic treatment with antidepressants directly affected astroglia, by increasing expression of a variety of receptors and transporters responsible for CNS homeostasis and limiting glutamate release [53, 60, 123, 171]. In conclusion, mood disorders are associated with astroglial degen eration and astroglial asthenia, which compromise brain homeostatic reserve and arguably synaptic transmission.

7.10.3. Addictive Disorders

Various nosological forms of addictive disorders are associated with astrogliopathies. Post-mortem analysis of the human brain samples revealed both astroglial reactivity with astroglial degeneration, and astroglial cell death with astroglial atrophy [9, 36, 72, 134, 145, 200, 225]. In the animal models of addiction with cocaine, methamphetamine and morphine, astroglial activation and increase in GFAP expression have been identified [25, 76, 84, 194]. In contrast, in the model of chronic alcoholism a decrease in GFAP expression and morphological atrophy of astrocytes were detected [75, 172]. In post-mortem tissues isolated from alcoholic sufferers, both hypertrophic GFAP positive astrocytes as well as areas with decreased GFAP expression and decreased density of astrocytes were described [52, 133].

The number of astrocytes is decreased in the prefrontal cortex of alcoholics [135]. A similar decrease in astroglial density and GFAP expression was detected in the prelimbic cortex of ethanol-preferring chronically alcoholic rats [133]. Additionally, a decrease in astrocyte density was observed in response to acute binge drinking in male (but not female) adult rats [111]. Ablation of astroglia with L-α-aminoadipic acid or uncoupling astroglial syncytia using a pharmacological inhibitor of connexin channels in the prefrontal cortex increases alcohol preference [132].

Addictive disorders are linked to astroglial plasmalemmal glutamate transport. Expression of EAAT2 as well as Sxc⁻ glutamate transporters is decreased in the context of alcoholism. Incidentally, total extracellular glutamate increases most likely due to an imbalance between glutamate uptake (EAAT2) and release (Sxc⁻) [141, 169, 170]. Increase in the expression of EAAT2 by treatment with β-lactam antibiotic ceftriaxone decreased alcohol dependence [161, 179].

7.11. Epilepsy

In epilepsy, astrocytes undergo substantial pathological remodelling, which greatly affects their homeostatic capabilities and is linked to pathophysiology of this disease. In particular, the epileptic astroglial phenotype includes changes (mutations and/or expression levels) in ion channels, receptors and transporters [19, 196]. Abnormal electrophysiological characteristics have been observed in astrocytes isolated from patients with mesial temporal lobe epilepsy and associated sclerosis. These astrocytes, in addition, have severe impairment of intercellular coupling [19]. Astrocytes in sclerotic tissue up-regulated the expression of GFAP, suggesting thus their activation. Decrease in K⁺ buffering seems to be the dominant feature of astroglial remodelling in epileptic brains, which results in an increase of extracellular K⁺ concentration [124, 140]. Such an increase in extracellular K⁺ can be sufficient to instigate seizures [206]. Abnormal astroglial K⁺ buffering, at least in part, is linked to a significant down-regulation of inward rectifier K_ir4.1 channels. Here, decreases in K_ir4.1 current density and protein content have been found in astrocytes from the human sclerotic CA1 hippocampal area [24, 93, 94]. Genetic deletion of KCNJ10 gene encoding K_ir4.1 channel specifically from astroglia resulted in impaired K⁺ buffering, depolarisation of astrocytes, motor impairments and early death [59]. Other studies confirmed this finding by demonstrating that deletion of K_ir4.1 channels induces epileptiform symptoms in animals [196]. Mutations of KCNJ10 gene in humans are associated with the development of SeSAME syndrome (also called EAST syndrome), an autosomal recessive disorder characterised by epilepsy, ataxia, sensorineural deafness, wasting renal tubulopathy, mental retardation and electrolyte imbalance [22, 182]. Whether the modifications of Na⁺/K⁺ ATPase (NKA), another critical component of astroglial K⁺ buffering (NKA is primarily responsible for K⁺ uptake, whereas K_ir4.1 channels for K⁺ release and shuttling back to neurones [29, 114]) contribute to SeSAME, it remains to be explored. One of the forms of migraine, the familial hemiplegic migraine type 2, is however associated with loss-of-function mutation of astroglial specific α2 subunits of NKA [39]. Considering fundamental similarities of pathogenesis of migraine and epilepsy we may expect some abnormalities of astroglial NKA in the later pathology.

Epileptic astrocytes also demonstrate compromised glutamate uptake and homeostasis [51]. Deletion of the astroglial EAAT2 glutamate transporter results in an epileptiform phenotype with lethal spontaneous seizures, increased susceptibility to acute cortical injury and seizures after administration of sub-convulsive doses of pentylenetetrazole [202]. Similarly, seizures and epileptiform phenotype were triggered by pharmacological inhibition of EAAT by intracerebroventricular injections of DL-threo-beta-benzyloxyaspartate [58]. Down-regulation of glutamine synthetase was also linked to epilepsy through affecting inhibition in neuronal networks [151]. Animals subjected to long-lasting pharmacological blockade of glutamine synthetase demonstrated seizures [20, 224], whereas levels of glutamine synthetase were found to be significantly decreased in the human hippocampus and amygdala of patients with temporal lobe epilepsy [64]. Finally, loss-of-function mutations of glutamine synthetase induced severe seizures [87]. Astrocytes can also contribute to pathogenesis of epilepsy through anomalous adenosine homeostasis, resulting from modified expression of the astroglia-specific adenosine kinase, which is the key enzyme for adenosine turnover in the CNS [10, 23]. Expression of adenosine kinase is high in tissues from subjects with pharmacologically refractory temporal lobe epilepsy [10, 11, 128]. Increase in expression and activity of adenosine kinase diminishes the availability of adenosine, thus increasing neuronal network excitability and increasing probability of seizures [117, 118].

7.12. Epilogue

Since the inception of neurobiology, we have had a conceptual roller coaster ride in regards to the role of neuroglia in pathology of the brain. Two centuries ago our founding fathers of gliology had a clear vision on the active role of glia, i.e. glia is more than putty and has prominent roles in pathophysiology of the brain. Awkwardly, the twentieth century brought a different view where starring role has been solely played by neurones. This dominant neurono-centric approach has been challenged by the resurgence of neurogliopathology in the past 20 years. While we here presented the astrocyte-centric view of the brain pathology, we surely support the notion that it is the interaction between neurones and glia that underlies physiology and pathology of the brain. These two major cellular constituents interact, so that perturbing one will affect the other. Thus, only intellectually acceptable approaches to grapple with the management of the brain diseases will be those that have gestalt assets.