Visual Defects and Ageing

Abstract

Many diseases are related to age, among these neurodegeneration is particularly important. Alzheimer’s disease Parkinson’s and Glaucoma have many common pathogenic events including oxidative damage, Mitochondrial dysfunction, endothelial alterations and changes in the visual field. These are well known in the case of glaucoma, less in the case of neurodegeneration of the brain. Many other molecular aspects are common, such as the role of endoplasmic reticulum autophagy and neuronal apoptosis while others have been neglected due to lack of space such as inflammatory cytokine or miRNA. Moreover, the loss of specific neuronal populations, the induction of similar mechanisms of cell injury and the deposition of protein aggregates in specific anatomical areas are very similar events between these diseases. Intracellular and/or extracellular accumulation of protein aggregates is a key feature of many neurodegenerative disorders. The existence of abnormal protein aggregates has been documented in the RGCs of glaucomatous patients such as the anomalous Tau protein or the β-amyloid accumulations. Intra-cell catabolic processes also appear to be common in both glaucoma and neurodegeneration. They also help us to understand how the basis between these diseases is common and how the visual aspects can be a serious problem for those who are affected.

Introduction

Health is defined by the World Health Organization as a state of complete physical, mental and social well-being and not just the absence of disease or infirmity. This state, which should be physiological, is undermined by ageing events. According to Flatt (2012), old age consists of age-progressive decline in intrinsic physiological function, involving tissues, organs and apparatuses, thus leading to an increase in age-specific mortality. Against this background, quality of life assumes significant implications from both the social and the economic points of view. Indeed, ageing combines all the main forms of neurodegenerative diseases that lead to irreversible damage to cognitive, motor and behavioral functions. Furthermore, proper cognitive functioning is essential to independent living and successful ageing (Valentijn et al. 2005).

In this article, we obviously cannot examine the cognitive functions of all the sensorineural organs that play a significant role in the quality of ageing. Nor can we consider all the diseases that impair the quality of life and of ageing. Rather, we will focus our attention on sight and ageing, with particular regard to visual-field defects and ocular ageing.

Eye Diseases and Their Impact on the Visual Field

Many individual features affect vision in ageing subjects; these are personal differences and environmental factors that may have an impact on the brain and ocular structures. For example, the density of the crystalline lens increases with age, although this condition does not constitute a true cataract (Xu et al. 1997), and pupillary miosis influences retinal illumination (Loewenfeld 1979). Although these are not true pathologies, they are able to affect spatial contrast sensitivity (Owsley 2011) and consequently also the visual field (Korth et al. 1989). One of the most serious diseases that involve vision and the eye is age-related macular degeneration (AMD), which is characterized by progressive degeneration of the retinal pigment epithelium and the photoreceptors immersed in it. The consequence of this disease is that the patient progressively loses central vision and with it the central visual field. AMD chiefly affects the cells targeted by the visual signal, rather than the output cells of the retina, i.e. the retinal ganglion cells (RGCs). In the eye, the photoreceptors are a hundred times more than ganglion cells. This implies that the visual signal generated by the photoreceptors is compressed and conveyed to the ganglion cells, and along the neurons to the visual cortex in the central nervous system (Meister 1996; Meister and Berry 1999). Therefore, although a visual field defect occurs in AMD, the structures affected are different from those involved in neurodegeneration or glaucoma. Indeed, in AMD, retrograde trans-synaptic degeneration of the retinal ganglion cell layer is one of the mechanisms contributing to permanent disability following visual post-geniculate pathway injury (Keller et al. 2014; Mitchell et al. 2015). Perimetry, a method for evaluating the spectrum of AMD severity, appears to be valid technique for assessing retinal sensitivity in AMD when colloid bodies/drusen >125 μm in diameter are present, but before the development of late AMD (Luu et al. 2013), while microperimetry provides information beyond that of visual acuity and contrast sensitivity in the functional assessment of AMD (Cassels et al. 2018).

However, numerous anatomic changes that occur in the eye with age are able to alter vision. In the aged eye, as well as in the brain, and therefore in neurodegenerative disease, normal antioxidant defense mechanisms decline, which increases the vulnerability of tissues to the effects of oxidative stress (Finkel and Holbrook 2000). For example, exposure to ultraviolet-A sunlight induces major modifications in the epithelial cells of the cornea (Zinflou and Rochette 2017), while in human corneal endothelial cells, a significant increase in DNA oxidative damage occurs (Joyce et al. 2009). In many studies, this type of damage has been assessed by measuring the levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), an indicator of oxidative DNA damage (Ames and Gold 1991). In the cell, when DNA is damaged events occur, which may include: DNA repair, cell cycle delay, entry into senescence, or induction of apoptosis (Toussaint et al. 2002; Zgheib et al. 2005). DNA-induced stress inhibits proliferation and induces cellular senescence by activating specific pathways (von Zglinicki et al. 2005), including those that cause intracellular oxidative stress, such as that of hydrogen peroxide (Erusalimsky and Skene 2009) or the mitochondrial damage that increases it (Agarwal and Sohal 1994). One of the indicators of the presence of senescent cells in tissues is senescence-associated-ß-galactosidase (SA-β-gal), although it should be noted that it is not specific, and that today several markers are used to identify senescent cells. For example, the changes in heterochromatin formation (Narita et al. 2003) or telomere length associated to DNA damage (Hewitt et al. 2012), or the presence of cyclin-dependent kinase inhibitors p21, p16 (Baker et al. 2016) have been used to type cells from ocular tissues. Moreover, it has been shown that senescent cells produce changes in metabolism, the epigenome and gene expression (de Magalhães and Passos 2017). Furthermore, these cells have a peculiar secretome profile, called Senescence-Associated Secretory Phenotype (SASP), which can affect the microenvironment of the tissues by modifying the production of growth factors, extracellular matrix (ECM) -degrading proteins and also pro-inflammatory cytokines and chemokines (Coppé et al. 2008). Many types of cells acquire senescent phenotypes with age: keratinocytes, endothelial cells, lymphocytes, smooth vascular muscle cells, and others (Rodier and Campisi 2011). This pattern is also seen in the pathogenesis of glaucoma, in which there is progressive decay of the trabecular meshwork and its cells, which are markedly aged (Saccà et al. 2016a, b). While senescence cannot cause proliferation loss in non-divisive cells, such as endothelial cells or neurons, these cells undergo major conversion to SASP (Salminen et al. 2011). Therefore, as their numbers dwindle, these cells lose their ability to proliferate and assume the SASP phenotype; moreover, subcellular damage occurs through cellular senescence, which results in tissue malfunction and then in macroscopically visible manifestations of ageing (Bhatia-Dey et al. 2016). In addition, the morphology of the optic nerve, retinal nerve fiber layer and maculae are reported to vary across racial groups and with age (Girkin et al. 2011). In humans, from 500 to 7000 axons are lost per year (Parikh et al. 2007), and a sharp decline in RNFL (Retinal Nerve Fiber Layer) occurs after 50 years of age, as has been observed in histological studies (Johnson et al. 1987). Furthermore, all tests of vision show a deterioration of performance with increasing age (Rudolph and Frisén 2007) as does the visual field test. Figure 14.1 shows the normal range of the visual field and of the Retinal Nerve Fiber Layer (NRFL). From what has been said so far, it emerges that old age, albeit not in itself a disease, is manifested as a functional decay that involves many factors, both physical, such as senile miosis or lens changes, and psychophysical, such as reflexes or perception. In older adults, Owsley (2016) has identified three visual features that can be seriously impaired by the development of common eye conditions and diseases of ageing. The first is “spatial contrast sensitivity”, where “contrast sensitivity” is the ability of the eye to recognize different shades of the same color in two adjacent areas. While normally very high, this sensitivity declines in some diseases affecting the transparency of the optical apparatus and in some diseases of the optic nerve. The second is “scotopic vision”; this is the kind of vision that enables us to see in poor light, and which corresponds to the ability of the photoreceptors to adapt to darkness. This function also declines with age. Moreover, older adults with severe dark adaptation delays are more likely to have several risk factors for AMD (Owsley et al. 2014). The third feature is “visual processing speed”; this refers to the speed at which visual information is processed automatically, i.e. without the subject having to focus intentionally on a specific visual target. This third visual function slows down with old age; this is one of the most inhibiting phenomena connected with human ageing (Birren and Fisher 1991), being related to activities such as driving a car or normal mobility.

Fig. 14.1

Normal optic nerve (left eye) (a) Normal visual field represented on a grayscale map. Darker areas indicate lower sensitivities, while lighter areas indicate higher sensitivities. This graphical representation allows field loss to be interpreted easily and is usually used to demonstrate vision changes to the patient. (b) Normal optic disc with a healthy neuro-retinal rim. (c) Retinal nerve fiber layer (RNFL) measured by optical coherence tomography over a 3.4-mm diameter circle centered on the optic nerve head. The green area is the 5th–95th percentile by age, the yellow area is the 1st–5th percentile, and the red area is below the 1st percentile. In this case, all the measurements (continuous black line) are in the green area

Globally, of the 7.33 billion people alive in 2015, an estimated 36.0 million were blind. Moreover, the number of people suffering from moderate or severe visual impairment increased from 159.9 million in 1990 to 216.6 million in 2015 (Bourne et al. 2017). Subjects aged 50 years and older account for 65% and 82% of the visually impaired and blind, respectively. The main causes of visual impairment are uncorrected refractive errors (43%) followed by cataract (33%); the leading cause of blindness is cataract (51%) (Pascolini and Mariotti 2012). Blindness caused by AMD is estimated at 5% (Zetterberg 2016), though AMD has become the most common cause of blindness in high-income countries (Bourne et al. 2014). In addition, diseases such as AMD and glaucoma have probably become relatively common causes of visual impairment and blindness worldwide (Pascolini and Mariotti 2012).

In this chapter, however, we will deal only with visual field defects that may occur during age-related illnesses, such as neurodegeneration and glaucoma, which have the same pathogenic root.

Visual Field and Neurodegeneration

The human eye is constantly exposed to sunlight and artificial lighting. Exogenous sources of ROS, such as UV light, visible light, ionizing radiation, chemotherapy, and environmental toxins contribute to oxidative damage to eye tissues. UV rays, besides being able to directly damage ocular tissue, can induce oxidative stress in irradiated cells through the production of ROS and riboflavin by activating tryptophan and porphyrin, which in turn can activate cellular oxygen (Ikehata and Ono 2011). Oxidative stress can be defined as an imbalance between the production of ROS and the antioxidant capacity of the cell. Long-term exposure to these exogenous sources of ROS puts the ageing eye at considerable risk, owing to the pathological consequences of oxidative stress. As all the ocular structures, from the tear film to the retina, undergo oxidative stress over time, the antioxidant defenses of each tissue are called upon to safeguard against degenerative ocular pathologies. The ocular surface and the cornea protect by the eyelid tissues but they are significantly exposed to oxidative stress of environmental origin (Saccà et al. 2013). Indeed, ultraviolet rays modulate the expression of antioxidants and proinflammatory mediators by interacting with corneal epithelial cells (Black et al. 2011). The decay of antioxidant defenses in these tissues is clinically manifested as pathologies, including pterygium (Balci et al. 2011), corneal dystrophy (Choi et al. 2009) and Fuch’s endothelial dystrophy (Jurkunas et al. 2010). The crystalline is highly susceptible to oxidative damage in ageing because its cells and intracellular proteins are not replaced, thus providing the basis for cataractogenesis. H₂O₂ is the main oxidant involved in the formation of cataracts and in damage to the DNA of the lens and membrane pump systems, thus leading to the loss of vitality of epithelial cells and death due to necrotic and apoptotic mechanisms (Spector 1995).

The trabecular meshwork is the anterior chamber (AC) tissue which allows the drainage of the aqueous humor. It is particularly susceptible to mitochondrial oxidative damage (Izzotti et al. 2009), which affects its endothelium. Its ensuing malfunction leads to an increase in intraocular pressure (IOP) and the onset of glaucoma (Saccà et al. 2016b).

As occurs in several neurodegenerative diseases, the normal antioxidant defense mechanisms of the eye decline with ageing; this increases the vulnerability of the eye to the deleterious effects of oxidative damage, as has also been seen with regard to the brain (Finkel and Holbrook 2000).

It is believed that mitochondrial free radicals are among the main causes of mitochondrial DNA damage (mtDNA). Several studies have found high levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of mtDNA oxidative damage, in the aged brain (Agarwal and Sohal 1994) and trabecular meshwork (Saccà and Izzotti 2014). High levels of 8-OHdG have also been found post-mortem in both the nuclear DNA (nDNA) and mtDNA of the brains of elderly subjects (Mecocci et al. 1993). Similarly, in glaucoma patients, damage to the trabecular mtDNA is greater than in healthy subjects. This increased sensitivity to mtDNA oxidative damage may be due to a lack of mtDNA repair mechanisms, lack of protection by the histone proteins, and the fact that mtDNA is located near the inner mitochondrial membrane, where reactive oxygen species are generated (Mecocci et al. 1993; Barja 2004). Neurodegeneration is characterized by a chronic and selective process of neuronal apoptosis and typically affects a specific neuronal system bilaterally. In Alzheimer’s disease (AD), for example, neurons in the medial temporal lobe and the limbic system are affected, while in Parkinson’s Disease (PD) the motor neurons are affected. In both cases, these neurons undergo apoptosis.

What patients with neurodegeneration have in common with glaucoma patients, in addition to old age, is neuronal loss, which leads to neurological changes in the visual field. In patients with AD, there is extensive loss of ganglion cells in the central retina (Blanks et al. 1996) and a volume reduction of the optic nerve (Kusbeci et al. 2015) (Fig. 14.2), where macular volume reduction reflects neuronal loss and is related to the severity of cognitive impairment (Iseri et al. 2006). Moreover, in patients with PD, the temporal retinal nerve fiber layer (RNFL) (Fig. 14.3) and central-parafoveal macular layers are reduced. Patients with greater RNFL damage tend to have more severe symptoms and, consequently, a lower quality of life (Mendoza-Santiesteban et al. 2017). The molecular mechanisms that determine neurodegeneration and diseases such as glaucoma and AMD are similar, in that they display the same pattern: first oxidative stress and inflammation, then mitochondrial dysfunction, and finally cellular apoptosis.

Fig. 14.2

Optic disc of a 67-year-old man affected by initial Alzheimer’s disease. The papilla appears paler than normal (a). The retinal nerve fiber layer (RNFL), measured by optical coherence tomography along a 3.4-mm diameter circle centered on the optic nerve head, shows a broad part below the 1st percentile (b)

Fig. 14.3

Optic nerve and visual field in a patient affected by Parkinson’s disease (left eye) (a) The visual field shows areas of decreased sensitivity. (b) The optic nerve head appears within normal limits. However, OCT measurement of the retinal nerve fiber layers (c) shows a significant reduction in the temporal sector. In neurological diseases, such as Parkinson’s disease and Alzheimer’s disease, visual field examination can only be performed in the early stages of the disease, as good collaboration on the part of the patient is necessary. Conversely, OCT can be performed even in more advanced stages, as the acquisition time is limited to a few seconds

These diseases have many other molecular aspects in common, such as autophagy; this is a metabolic process through which cells degrade cytoplasmic substrates through their lysosomes, and participates in the basal turnover of long-lived proteins and organelles. There are three different types of autophagy: macroautophagy, chaperone-mediated autophagy, and microautophagy, all of which are indispensable to cellular homeostasis (Plaza-Zabala et al. 2017). Indeed, dysfunctional mitochondria appear to be selectively removed by means of autophagy (Li et al. 2012). By contrast, non-selective autophagy occurs when mitochondria are digested by lysosomes to obtain nutrients and energy, and plays an important role in promoting neuronal health and survival (Plaza-Zabala et al. 2017). Mitochondria play a vital role in several phases of autophagy, from initial autogenous biogenesis and autophagic regulation through beclin-1 to autophagy-mediated cell death (Rubinsztein et al. 2012). In the normal human brain, autophagy diminishes with ageing (Rubinsztein et al. 2011). Indeed, insufficient protective autophagy accelerates both ageing and AD pathology, and is possibly caused by defects in autophagosome fusion with lysosomes (Barnett and Brewer 2011). In the nervous system, autophagy is associated with the maintenance of the normal balance between the formation and degradation of cellular proteins. As neurons are susceptible to the accumulation of aggregated or damaged cytosolic compounds or membranes, their survival depends on autophagy (Tooze and Schiavo 2008). Autophagy can inhibit proinflammatory signaling by eliminating dysfunctional mitochondria (Saitoh and Akira 2010) and can also inhibit the activation of the NLRP3 inflammasome – the caspase-1 activation complex required for the production of interleukin-1β – by removing permeabilizing or ROS-producing mitochondria (Zhou et al. 2011). These anti-inflammatory effects of autophagy are useful both to brain health and in neurodegenerative processes and pathological ageing, which are often accompanied by chronic inflammation (Rubinsztein et al. 2011). Mutations in Presenilin-1, which is a protein of the gamma secretase complex, and which plays an important role in the generation of amyloid beta, are associated with the onset of AD, reducing lysosomal acidification and inducing a blockade of autophagy flow in the fibroblasts of AD patients (Lee et al. 2010). Phenotypes indicative of defective autophagy are also found in PD, in which protein aggregation and mitochondrial damage occurs (Kim et al. 2017). Moreover, the native form of α-Synuclein, which is a presynaptic neuronal protein involved in PD pathogenesis, is degraded by means of chaperone-mediated autophagy (Cuervo et al. 2004). Thus, autophagy plays an important role in cellular homeostasis in neurodegenerative diseases, too. Moreover, mitochondria participate in the formation of autophosomes; indeed, the membranes of these organelles are transiently shared (Hailey et al. 2010). The main factors influencing autophagy are the perturbation and starvation of Ca⁺⁺ homeostasis. Signals from Ca⁺⁺ perturbations might be integrated into the unfolded protein response (UPR) and the activation of endoplasmic reticulum (ER) stress (Kania et al. 2015). In PD, prolonged stimulation of mitochondrial oxidative phosphorylation occurs, owing to an excess of Ca⁺⁺; this causes oxidative stress, which impairs mitochondrial function, increases mitophagy and induces high α-synuclein expression (Surmeier et al. 2016). Ca⁺⁺ dysregulation may involve several mechanisms, since the ER, the lysosomes and the Golgi apparatus, as well as the mitochondria, are important intracellular Ca⁺⁺ reservoirs (Patel and Muallem 2011; Kilpatrick et al. 2013). An accumulation of unfolded or misfolded proteins in the ER leads to stress conditions. To mitigate such circumstances, stressed cells activate a homeostatic intracellular signaling network to restore normal cell function by halting protein translation, degrading misfolded proteins, and activating the signaling pathways that lead to the increased production of the molecular chaperones involved in protein folding. If these molecular conditions are not restored, the UPR is directed towards apoptosis. Although ER stress and autophagy can function independently, they are dynamically interconnected and ER stress can either stimulate or inhibit autophagy (Rashid et al. 2015). ER stress can also promote the NF-kB activation associated to inflammatory pathways (Muriach et al. 2014) and activate cellular inflammatory pathways, which, in turn impair cellular functions and induce mitochondrial changes and, finally, cell apoptosis (Rao et al. 2004). ER stress also causes cellular accumulation of the ROS associated to oxidative stress (Cullinan and Diehl 2006), which, in turn, can reciprocally promote ER stress, thus creating a vicious circle.

Visual Field and Glaucoma

Glaucoma is a neurodegenerative disease characterized by progressive optic atrophy, which is the result of the death of retinal ganglion cells due to apoptosis induced by the inhibition of cell survival. This disease causes visual field alterations, and can be acute or chronic, depending on whether the corneal iris angle is open or closed. The opening of this angle enables the aqueous humor (AH) to circulate. Produced by the ciliary body, the AH passes through the pupil into the AC and flows through the trabecular meshwork (TM).

Trabecular Meshwork Organization

For a long time it was believed that AH outflow was a passive phenomenon, and that the TM was a structure similar to a filter. This is not so. The TM consists of endothelial cells that are immersed in their matrix to form a network of beams, in which the matrix occupies the spaces between the beams (Tian et al. 2000) (Fig. 14.4). TM cells respond to mechanical stretching by increasing ECM turnover and reducing total versican mRNA levels (Keller et al. 2007). As is obvious, AH outflow is an active phenomenon, and is regulated by the cells of the TM. Indeed, the TM is a true organ made up of endothelial cells that are able to increase or reduce AH outflow in accordance with the requirements of the eye (Fig. 14.5). The TM incorporates two barriers: the first is constituted by trabecular meshwork endothelial cells that are immersed in the AH of the AC; the second is formed by the endothelial cells that line the anterior wall of Schlemm’s canal (SC). The first controls the second through a cytokine system, which modulates the permeability of the endothelial cells of the SC (Alvarado et al. 2005a). These cytokines include IL1a, IL1b and IL8 (Alvarado and Shifera 2010). In reality, interactions between these barriers occur in both directions and involve relationships within both barriers. Indeed, the endothelial cells lining the SC, which make up the second barrier, are juxtaposed to juxtacanalicular tissue (JCT); these cells form tight junctions, providing a significant barrier to the passage of fluid (Gong et al. 2001, 2002). They also act as a “control” site, regulating the outflow of AH from the eye by increasing/reducing the permeability of this barrier, and drive a mechanism controlling SC endothelium permeability (Alvarado et al. 2005a, b). By altering the physiological catabolism of the ECM, TM cells are able to change the TM outflow, and hence to regulate aqueous outflow resistance (Aga et al. 2014). In this structure, endothelial cells are organized on the collagen beams, maintaining evident cell–ECM focal contact-like structures and adherent-type cell–cell junctions (Tian et al. 2000). The cytoskeletal interactions between JCT and the SC can be regulated by a variety of environmental and cytoplasmic factors, such as the level of extracellular calcium, activation of specific small G-proteins, mechanical tension and hydrostatic pressure (Ye et al.1997), and particular molecular components of tight junctions may help to regulate flow resistance (Underwood et al. 1999). The spaces between JCT cells and ECM fibers contain a ground substance consisting of various proteoglycans and hyaluronan (Gong et al. 1992; Tamm 2009). Therefore, the endothelial cells that form this outflow pathway, besides having the ability to secrete proteins and cytokines, have a cytoskeleton that allows them to change shape and, accordingly, their gene expression (Saccà et al. 2016b). This plasticity is also due to the contractile properties of the ciliary body and the trabecular meshwork; according to the circumstances, these properties are activated through various signal transduction pathways involved in the regulation of smooth muscle contractility (Wiederholt et al. 2000). The TM is thought to be a smooth muscle-like tissue with contractile properties (Lepple-Wienhues et al. 1991). Contraction and relaxation of the cells are thought to regulate aqueous humor outflow (Wiederholt 1998). Indeed, the TM possesses smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure; the TM and ciliary muscle appear to be functional antagonists, with ciliary muscle contraction leading to distension of the TM and subsequent reduction in outflow, and with TM contraction leading to the opposite effect (Wiederholt et al. 2000). Similarly, increases or decreases in the volume of TM cells could influence outflow (O’Donnell et al. 1995; Soto et al. 2004). TM cell volume is influenced by the activities of ions (Al-Aswad et al. 1999; Mitchell et al. 2002). Moreover, it is thought that both cellular contractile mechanisms and cell volume regulatory mechanisms are functionally linked (Dismuke et al. 2008), and may be part of the homeostatic mechanisms of the TM whereby outflow is regulated. Furthermore the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel has been shown to regulate TM cell volume and contractility (Wiederholt et al. 2000; Soto et al. 2004) and outflow facility (Soto et al. 2004). The flow from the AC to Schlemm’s canal takes place basically through two pathways: a paracellular route through the junctions formed between the endothelial cells (Epstein and Rohen 1991), and a transcellular pathway through intracellular pores (Johnson and Erickson 2000) or giant vacuoles in the cells themselves. SC cells probably have the ability to modulate local pore density and the filtration characteristics of the inner-wall endothelium on the basis of local biomechanical cues (Braakman et al. 2014).

Fig. 14.4

(a, b) TM cells were fibronectin-coated under standard culture conditions. Images were generated by means of a GE In Cell 1000 high-content imaging system and were colored for nuclei (blue), actin fibers (red), and focal adhesions (green). As DNA is stained blue, the large clumps of blue just above center are cell nuclei. The red lines are filaments of actin extending throughout the cell, while the green patches at their tips are focal adhesions. These images document the plasticity of these endothelial cells, and the different shapes of the same cell-type demonstrate that these cells have the ability to change shape; the endothelial cells are endowed with an intricate actin network, which extends from the cell wall throughout the cell body to the nucleolus (blue) and enables these cells to assume various shapes. Owing to the functions of their unique and complex metabolism, these endothelial cells are able to change their gene expression in order to preserve their barrier function of the TM. (Saccà et al. 2016b)

Fig. 14.5

(a) The conventional aqueous outflow pathway: from the ciliary process the aqueous humor flows through the pupil into the anterior chamber, where it encounters the trabecular meshwork endothelial cells that line aqueous channels, and then subsequently encounters the endothelial cells that line the lumen of Schlemm’s canal. (b) Anatomy of the iridocorneal angle with the structures observable on gonioscopy. Schlemm’s canal is not normally visible. (c) The endothelial cells that delineate the structures of the collagen framework of the trabecular meshwork are equipped with a cytoskeleton and thus are able to change their shape. The cytoskeleton (red filaments) is attached to the nuclear membrane (colored in blue) and can, in milliseconds, send signals to the nucleus in order to alter the expression of genes in an attempt to adapt to biomechanical injury. (d) The proteins released by the trabecular-toothed cells in the anterior chamber may become pro-apoptotic signals for the retina, and in particular for retinal ganglion cells, activating apoptosis. (e) If glaucoma is not adequately treated, the typical appearance of optic nerve head atrophy, with its characteristic cupping, is observed

Factors Involved in Glaucoma Pathogenesis

Numerous factors, both metabolic and environmental, can affect the conventional outflow pathway, the main one being its cellularity. Indeed, the decline in the cellularity of human TM endothelial cells is linearly related to age, and plays a major role in glaucoma pathogenesis (Alvarado et al. 1981, 1984). When the capacity of the TM cells to remove aqueous humor decreases, IOP increases (Saccà and Izzotti 2014). The relationship between IOP and optic nerve damage has not yet been elucidated, but it is certain that this relationship is real. Indeed, the only currently recognized anti-glaucoma therapy is that which lowers IOP (Konstas et al. 2015). Therefore, all that is able to influence the functioning of the endothelial cells plays a role in the pathogenetic cascade of glaucoma. Oxidative damage is now recognized as an important step in the pathogenesis of glaucoma. Indeed, it affects the TM cells, and if it is measured in terms of 8-hydroxy-2′-deoxyguanosine (8-OH-dG) levels, the DNA of TM cells in glaucoma patients is seen to be altered in comparison with the healthy TM; moreover, this damage is directly proportional to the IOP level and to the visual field damage (Saccà et al. 2005). Both in ageing and during the course of glaucoma, normal antioxidant defenses decline, which determines an increased susceptibility to the effects of this type of damage (Finkel and Holbrook 2000) The TM is the tissue most sensitive to oxidative radicals in the AC (Izzotti et al. 2009). As in other neurodegenerative diseases, mitochondrial involvement is also seen in glaucoma; in particular, mtDNA damage occurs in the target tissue of POAG, the TM. Such damage is detectable only in the TM, and not in other AC tissues, cornea or iris (Izzotti et al. 2010b). The dysfunction of oxidative phosphorylation secondary to mtDNA mutations can reduce ATP production and the generation of reactive oxygen (ROS). An increase in ROS that exceeds the antioxidant capacity of the tissue results in oxidative stress, contributing to the ageing process through the induction and further progression of cellular senescence. The defective mitochondrial function in the TM cells of patients with POAG renders these cells abnormally vulnerable to Ca²⁺ stress, with subsequent failure of IOP control (He et al. 2008). Conversely, the increased expression of SIRT1 antagonizes the development of oxidative stress-induced premature senescence in human endothelial cells (Ota et al. 2007). The sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases that help to regulate the lifespan of several organisms and may provide protection against diseases related to oxidative stress-induced ocular damage (Mimura et al. 2013). In the case of glaucoma, this is likely to occur through the interaction of SIRT1 with eNOS (Ota et al. 2010). Indeed, eNOS activity in HTM cells regulates inflow and outflow pathways (Coca-Prados and Ghosh 2008), and the regulation of eNOS is, in turn, influenced by the activation of Rho GTPase signaling (Shiga et al. 2005) in the AH outflow pathway, which influences actomyosin assembly, cell adhesive interactions and the expression of ECM proteins and cytokines in TM cells in a cascade-like manner (Zhang et al. 2008). It is evident therefore that the mitochondria of TM cells play a crucial role in meeting the high metabolic demand of these cells. Mitochondrial integrity declines with age, aged organelles being morphologically altered and producing more oxidants and less ATP than younger organelles (Shigenaga et al. 1994). The mitochondrial dysfunction that occurs during the course of glaucoma remains of unknown origin. However it is known that mitochondrial DNA deletion, which is correlated with levels of 8-OH-dG (Hayakawa et al. 1992), is far greater in the TM of patients with glaucoma than in controls. This finding is paralleled by a decrease in the number of mitochondria per cell and by cell loss (Izzotti et al. 2010b), with the result that mitochondria bearing this deletion are less efficient in ATP production and release more ROS than intact mitochondria (Izzotti 2009), thereby causing an energy deficit and tissue atrophy (Morris 1990). Moreover, the cytoskeleton-dependent changes in ATP release are correlated with changes in cell volume regulation (Sanderson et al. 2014), and the entire TM cell homeostasis is altered. In glaucomatous TM tissue, the increased number of cells positively stained for senescence-associated-β-galactosidase (SA-β-Gal) activity has been seen to disrupt the local tissue micro-environment through the over-expression of several pro-inflammatory cytokines and the production of ROS (Liton et al. 2005). Oxidative DNA damage in the TM has been significantly correlated with age, and there is a significant relationship between DNA oxidative damage and autophagy activation (Pulliero et al. 2014). Autophagy is a catabolic process involving lysosomal degradation, which has the characteristic of activating a cellular survival mechanism against a variety of stressors. During glaucoma, the TM cells react to IOP increase by triggering autophagy (Porter et al. 2013) even though dysregulation of the autophagic pathway in TM cells occurs (Porter et al. 2015). However, the activation of autophagy must be coupled with the lysosomal system; indeed, concurrently with autophagy, the endoplasmic reticulum (ER) undergoes stress in HTMcs (Li et al. 2011).

Myocilin and Optineurin

Myocilin may play a role in autophagy. Myocilin is a glycoprotein that is normally expressed in both ocular and non-ocular tissues. Although its physiological function is as yet unknown, it may play a role in the development of retinal cell apoptosis (Koch et al. 2014) and in axonal myelination and oligodendrocyte differentiation (Kwon et al. 2014). This protein is assembled in the extracellular space between the ER and Golgi complex through the secretory pathway that is responsible for the synthesis, folding and delivery of cellular proteins; here, it may undergo mutation, leading to myocilin aggregation, ER stress and TM cell toxicity (Stothert et al. 2016). Indeed, the chronic accumulation of misfolded proteins in the ER can facilitate cell death (Gorbatyuk and Gorbatyuk 2013; Zhu and Lee 2014). Furthermore, autophagy contributes to cellular homeostasis through the turnover of mitochondria, ER and peroxisomes (Johansen and Lamark 2011; Wang and Klionsky 2011). Myocilin mutations are typically associated with high IOP; for this reason they have more impact on the HTM cells, while optineurin (OPTN) mutations are associated with normal-tension glaucoma, even though its expression is increased by IOP increases (Vittow and Borras 2002). OPTN plays an important role in regulating several genes, including myocilin (Park et al. 2007). Moreover, OPTN expression is regulated by various cytokines, particularly NF-κB, which can be activated by increased IOP, ageing, vascular diseases and oxidative stress (Saccà and Izzotti 2014). Optineurin can also mediate the removal of protein aggregates through a ubiquitin-independent mechanism that also serves as a substrate for autophagic degradation (Ying and Yue 2016). Optineurin is therefore involved not only in glaucoma but also in other neurodegenerative diseases or in cancer, and again, in age-related macular degeneration (Wang et al. 2014). Myocilin mutations in the MYOC gene are observed in 10–30% of cases of juvenile-onset open-angle glaucoma (Shimizu et al. 2000), and in 2–4% of patients with POAG (Stone et al. 1997). In combination, mutated myocilins may exacerbate sensitivity to oxidative stress (Joe and Tomarev 2010). Furthermore, it is interesting that over-expession of the senescence-related biomarkers SM22 and osteonectin (SPARC) occurs in conditions of oxidative stress. Both proteins are involved in ECM turnover (Nair et al. 2006; Dumont et al. 2000). SPARC over-expression increases IOP in perfused cadaveric human anterior segments as a result of a qualitative change in the juxtacanalicular tissue ECM (Oh et al. 2013). Furthermore, the function of myocilin as an extracellular protein may link ECM molecules such as fibronectin and laminin with matricellular proteins such as SPARC (Aroca-Aguilar et al. 2011). SM22 is an actin-binding protein involved in senescence-associated morphological changes (Nair et al. 2006). Increasing macroautophagy can delay the ageing process and extend tissue longevity (Stothert et al. 2016). In glaucomatous TM cells, the autophagic mechanism is dysregulated, and they show a decreased response to oxidative stress (Porter et al. 2015). Micro RNAs (miRNAs), post-trascriptional regulators of gene expression, are associated with the development of stress-induced premature senescence (SIPS) in the HTM. TGF-β also induces SA-β-Gal activity and increases the miRNA levels of senescence-associated genes (Frippiat et al. 2001), thereby promoting SIPS. The down-regulation of members of the miR-15 and miR-106b families may contribute to some features of senescent cells, such as increased resistance to apoptosis; likewise, the up-regulation of miRNAs, such as miR-182, may contribute to specific changes in gene expression that are associated with the senescence phenotype (Li et al. 2009). It is evident that this situation leads to a functional decay of the TM, in which both oxidative stress and mitochondrial dysfunction induce endothelium dysfunction of trabecular cells (Saccà et al. 2016a). The endothelium of the TM is immersed in the aqueous humor, and its operation is comparable to that of a small vessel. Indeed, the endothelial leukocyte adhesion molecule-1 (ELAM-1) is found in the AH; this oxidatively induced molecule is known as an early marker of atherosclerotic plaque in the vasculature (Eriksson et al. 2001), along with all the other atherosclerotic biomarkers (Saccà et al. 2012).

The Proteome

In the glaucomatous aqueous humor, the proteome is altered and has special characteristics: many proteins expressed at high levels in healthy subjects are reduced in POAG patients; by contrast, other proteins detected at low levels in normal aqueous humor are increased in glaucoma (Table 14.1). Total proteins do not show quantitative differences between POAG patients and controls; the qualitative differences, however, are significant (Izzotti et al. 2010a). These differences can be divided into six groups, the first of which concerns mitochondria, mitochondrial proteins involved in the electron transport chain, trans-membrane transport, protein repair, and the maintenance of mitochondrial integrity. The proteins involved are found in the AH because they come from mitochondria that no longer work, and also because trabecular cells that die release their contents into the medium. The apoptosis that occurs in ocular tissues during POAG is induced by a variety of mechanisms: mainly mitochondrial damage, but also inflammation, vascular dysregulation, and hypoxia. It is evident that, as the cells gradually undergo apoptosis, their physiological TM functions fail. The barrier function is altered, and this is reflected in the AH by the presence of a group of proteins associated with cell adhesion defects. This group includes chains, junction proteins and cadherins. All the biological events observed in glaucoma are indicative of the progressive functional failure of the TM. Indeed, the glaucomatous AH contains another important group of proteins: those that induce apoptosis. One of these proteins, which seems to be very important and which is expressed more than in healthy subjects, is insulin receptor substrate 1 (IRS-1). This catalyzes the intramolecular autophosphorylation of specific tyrosine residues of the insulin β subunit, further enhancing the binding of tyrosine kinase to the receptor for other protein substrates (White et al. 1985). IRS-1 exerts an important biological function both in metabolic and mitogenic pathways and in signal-activating pathways, including the PI3K pathway and the MAP kinase pathway. It should be remembered that the PI3K gene is related to the system of reporting insulin/IGF-1, one of the mechanisms of regulation of ageing tissue and that the PI3K/AKT/mTOR pathway is an intracellular signaling pathway involved in apoptosis. Indeed, in vascular tissue, activation of the PI3K/Akt/mTOR survival signal pathway and concomitant suppression of the p38 MAPK proapoptotic pathway protects the endothelium against damage caused by oxidative stress, cell migration and/or proliferation and apoptosis/survival (Joshi et al. 2005). The MAPK (originally called ERK) pathway is a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. The pathway includes many proteins, which communicate by adding phosphate groups to a neighboring protein, thereby acting as an “on”/“off” switch. When one of the proteins in the pathway is mutated, it can be stuck in the “on” or “off” position, which is a necessary step in the development of many diseases. During glaucoma, the components of this MAP kinase pathway in the TM are dramatically affected by TNF-α, and inhibition of extracellular signal-regulated kinase phosphorylation blocks changes in MMP and tissue inhibitor expression (Alexander and Acott 2003). Furthermore, the mitogen-activated protein MAP kinase superfamily constitutes a signaling pathway that is also active in the posterior segment. Indeed, p38, a member of this superfamily, is involved in the RGC apoptosis that is mediated by glutamate neurotoxicity through NMDA receptors after damage to the optic nerve (Kikuchi et al. 2000). Again, the MAP signaling pathway is probably involved in the induction and/or maintenance of the activated glial phenotype in glaucoma. Because MAPKs are involved in determining the ultimate fate of these cells, their differential activity in neuronal and activated glial cells in the glaucomatous retina may be partly associated with the differential susceptibility of these cell types to glaucomatous injury (Tezel et al. 2003). It is interesting that apoptosis signal-regulating kinase 1 is a MAP kinase involved in neural cell apoptosis after various kinds of oxidative stress (Harada et al. 2006) and its deficiency attenuates neural cell death in normal-tension glaucoma-like pathology in both neural and glial cells, in which the TNF-induced activation of p38 MAPK is suppressed and inducible nitric oxide synthase is produced (Harada et al. 2010). The PI3K pathway also modulates the expression of angiogenic factors, such as vascular endothelial growth factor, nitric oxide and angiopoietins (Karar and Maity 2011). Moreover, IRS-1 plays necessary roles in insulin-signaling pathways leading to the activation of eNOS Ca²⁺-mediated pathways (Montagnani et al. 2002). Gogg et al. (2009) introduced the concept of “selective” insulin resistance, involving IRS-1 and the PI3kinase pathway, as an underlying factor for the dysregulation of microvascular endothelium, in which MAPK are involved in increasing endothelin (ET)-1levels. This could happen at the molecular level in the TM. Indeed, the levels of pro-inflammatory cytokine TNF-alpha are reported to be significantly higher in the glaucomatous AH (Sawada et al. 2010). TNF-alpha induces a state of insulin resistance in terms of glucose uptake in myocytes because of the activation of pro-inflammatory pathways that impair insulin signaling at the level of the IRS proteins (Lorenzo et al. 2008). This increases contractile dysfunction (Reid and Moylan 2011; Bhatnagar et al. 2010) and mitochondrial ROS, which in turn activate apoptosis signal-regulating kinase 1, which aggravates endothelial cell dysfunction. It is also possible that the diffusion of these proteins in the posterior segment, through the route described by Smith et al. (1986) may reduce retinal Müller cell death (Walker et al. 2012).

Table 14.1 POAG-marker proteins in aqueuous humour as detected by Ab microarray (Izzotti et al. 2010a)

Another important group of proteins in the glaucomatous aqueous humour that reflects TM motility damage is that of Protein Kinases (PKC). Indeed, PKC plays an important role both in the regulation of phosphorylation of myosin light chain, which induces cellular contraction and in the dynamics of the cytoskeleton in the Trabecular Meshwork. Furthermore, PKC could affect outflow, by influencing the cell shape (expansion, contraction and morphological changes) in the trabecular and sclerocorneal cells. This group also includes one of the proteins most abundantly expressed in the AH of POAG, i.e. A kinase (PRKA) anchor protein 2. Protein phosphorylation is one of the most important mechanisms of enzyme regulation and signal transduction in eukaryotic cells. cAMP-dependent protein kinase (PKA), one of the first protein kinases discovered, appears to be the main ‘read-out’ for cAMP to downstream signaling pathways. These downstream substrates include other protein kinases, protein phosphatases, other enzymes and ion channels. Metabolism, gene transcription, ion channel conductivity, cell growth, cell division and actin cytoskeleton rearrangements are modulated by PKA-catalyzed phosphorylation in response to hormonal stimuli (Francis and Corbin 1994; Scott 1991). Localization of the cAMP- PKA and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). These AKAPs are classified on the basis of their ability to associate with the PKA holoenzyme inside cells (Colledge and Scott 1999); they function as targeting units and tether the kinase to specific subcellular localizations (Huang et al. 1997). The AKAPs are a group of structurally diverse proteins, which have the common function of binding to one or more of the regulatory subunits of cAMP-dependent protein kinase A (PRKA) and confining the holoenzyme to discrete locations within the cell. Activation of PRKA usually results from the binding of cAMP to the R subunits of PRKA, this promotes dissociation and activation of the catalytic subunits, leading to a wide variety of cellular responses. The encoded protein is expressed in endothelial cells, cultured fibroblasts, and osteosarcoma cells. It associates with protein kinases A and C and phosphatase, and serves as a scaffold protein in signal transduction. Cytoskeletal signalling complexes facilitate this process by optimizing the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. AKAP mediated organization of kinases and phosphatases is particularly important for the transduction of signals to the cytoskeleton (Diviani and Scott 2001). Indeed, AKAP-Lbc is a protein kinase A-anchoring protein that also functions as a scaffolding protein to coordinate a Rho signaling pathway (Diviani et al. 2001). The Rho family of small GTPases are the principal transmitters of signals from transmembrane receptors that stimulate actin filament nucleation (Bishop and Hall 2000). The actin cytoskeleton is critical to a variety of essential biological processes in all eukaryotic cells, including the establishment of cell shape and polarity, motility, and cell division (Hall 1998). PRKA activation has been shown to negatively regulate RhoA signaling (Lang et al. 1996; Chen et al. 2005).These signals influence fundamental cell properties such as shape, movement and division (Scott 2003). PRKA 2 is highly enriched in mitochondria (Wang et al. 2001). This protein is a cell growth-related protein. It is well known that from anatomical, physiological and pathological perspectives, the Anterior Chamber (AC) is similar to a vessel and behaves like a vessel, so much so that during the course of POAG, the AC is characterized by mechanisms and molecular events resembling those that occur during atherosclerosis (Saccà et al. 2012). The presence of PRKA2 in AH of the AC is justified on the basis of the endothelial dysfunction that occurs during POAG (Saccà and Izzotti 2014). From a patho-physiological point of view, we think that this is a result of the reduction in TM endothelial cells that occurs in glaucoma (Alvarado et al. 1984), due both to ageing (Alvarado et al. 1981) and to mitochondrial dysfunction (Izzotti et al. 2010b, 2011). Probably, protein which originates from the cytoplasm of endothelial cell is lost into AH, where it acts as a signal to the surviving cells or other tissues. Indeed, oxidative stress is a plausible mechanism for the development of glaucoma, manifesting its effects on the HTM (Izzotti et al. 2003) as an IOP increase (Saccá et al. 2005).The TM endothelium is involved in modulating the permeability of the endothelial barrier and the release of endothelins and nitric oxide. In AH, there are many factors that have a protective role on endothelial cells, such as GSH, which protects anterior segment tissues from high levels of H₂O₂ (Costarides et al. 1991). Unfortunately, the TM is highly susceptible to oxidative injury (Izzotti et al. 2009) so the increased concentration of NO, that occurs in glaucoma (Tsai et al. 2002) reacts with anion superoxide to form peroxynitrite. The production of peroxynitrite is counteracted by the antioxidant defenses and repair systems located in the AC tissues and AH (Saccà et al. 2007). Together, however, NO and peroxynitrite can suppress eNOS expression via the activation of RhoA, hence causing vascular dysfunction (El-Remessy et al. 2010). Moreover, myocilin, which is localized in TM cells (Tamm 2002), has several functions that are mediated by Rho GTPase and cAMP/protein kinase A signaling. Indeed, when moderately over-expressed, myocilin induces a loss of actin stress fibers and focal adhesions (Wentz-Hunter et al. 2004), inhibits the adhesion of human TM cells to ECM proteins, and compromises TM cell-matrix cohesiveness leading to TM damage (Shen et al. 2008). In addition to acting on the motility of the trabecular meshwork, it should be stressed that the AKAP 2 regulates many ion channels, including those of Na⁺ involving PCK (Bengrine et al. 2007) and K⁺ channels (Zhang and Shapiro 2012). The TM cells utilize Na-K-Cl cotransport to modulate their intracellular volume, and thus the volume of the paracellular pathways through which aqueous humor may travel (Brandt and O’Donnell 1999). Thus, the role of AKAP 2 could also be related to the maintenance of the endothelial barrier within the anterior chamber. In the course of glaucoma endothelial dysfunction occurs which impairs the functioning of this barrier (Saccà et al. 2015).

Another important protein that is a highly expressed by human endothelial trabecular meshwork cells is the Actin related protein 2/3 complex, subunit 3, 21 kDa (Arp2/3). Human Arp2 and Arp3 are very similar and this protein complex has been implicated in the control of actin polymerization in cells (Welch et al. 1997). Moreover, the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion (Machesky et al. 1997). It is interesting that the Arp2/3 complex is critical for spine and synapse formation in the central nervous system, and this synaptic plasticity, which underlies cognitive functions such as learning and memory is attributed to the reorganization of actin (Wegner et al. 2008). In all probability we discovered the Arp2/3 complex in glaucomatous AH because the TM endothelial barrier is altered during this disease (Saccà et al. 2012), likely owing to oxidative stress (Saccà et al. 2009). Indeed, actin cytoskeleton rearrangements are the basis of many fundamental processes of cell biology such as motility, adhesion, mitosis, endocytosis, and morphogenesis (Moreau et al. 2003). Actin not only facilitates membrane deformation, cytoskeleton remodeling and the formation of vesicles, but also contributes to vesicle movement and targeting within the cell. Indeed, normal regulation of vesicular transport events is essential to cell proliferation and apoptosis, as well as to the maintenance of homeostasis. Deregulation of vesicular transport can lead to decreased capacitive calcium entry, which in turn results in cell apoptosis (Jayadev et al. 1999). Rho GTPases play a key role in vesicle trafficking through their ability to regulate the actin cytoskeleton (Cory et al. 2003). Actin plays multiple roles in vesicle trafficking (Smythe and Ayscough 2006; Lanzetti 2007). Indeed the Arp2/3 complex leads to actin polymerization (Chi et al. 2013). Furthermore, actin assembly regulates the nuclear import of Junction-mediating and regulatory protein which is a regulator of both transcription and actin filament assembly in response to DNA damage (Zuchero et al. 2012).

During glaucoma TM motility is altered (Khurana et al. 2003) and the presence of the Arp 2/3 complex, together with other classes of AH proteins (in the AH) could represent an attempt to restore normal TM motility. This hypothesis fits well with the finding that Protein kinase C levels are significantly increased in the AH of POAG (Izzotti et al. 2010a). Moreover, the presence of Arp 2/3 complex in POAG AH indicates that the endothelium of the TM behaves as a vascular tissue and therefore may change shape. The results obtained by Mao et al. (2011) implicate podosomes in normal development of the iridocorneal angle and the genes influencing podosomes as candidates in glaucoma. Moreover, in various models, the cytoskeletal dynamics underlying these processes have been shown to be driven by small G-protein members of the Rho family. Indeed, endothelin-1 (ET-1) is known to induce Ca²⁺-independent contraction of the trabecular meshwork. This contraction involves RhoA and its kinases as intracellular mediators (Renieri et al. 2008). Protein kinase C (PKC) levels are significantly increased in POAG AH influencing cytoskeletal dynamics within the TM (Izzotti et al. 2010a). PCK activation triggers other regulatory mechanisms which in turn influence the shape of TM cells (Khurana et al. 2003).

Importantly, Rho GTPases regulate actin dynamics by acting as molecular switches that transduce signals from activated membrane receptors to cytoskeleton organizers (Van Aelst and D’Souza-Schorey 1997). Podosomes in the machinery required for actin polymerization constitute the localization dedicated to a specific physiological process such as angiogenesis, or vascular permeability, or represent the manifestation of a pathological status, with inevitable consequences on endothelial cell functions (Moreau et al. 2003). In addition, it is important to remember that TM cells appear to sense an actomyosin-derived contractile force and induce ECM synthesis/assembly via Rho GTPase activation (Zhang et al. 2008). Another group of proteins present in the glaucomatous aqueous humor is that of the proteins involved in oxidative stress; in which the reduced expression of antioxidant enzymes SOD and GST worsens the imbalance between ROS and NOS that occurs during the course of glaucoma (Bagnis et al. 2012), aggravating molecular damage.

Lastly, another group of important proteins is that of neuronal proteins. Nestin and the above mentioned Optineurin belong to this group. Nestin, an intermediate filament protein, is thought to be expressed exclusively by neural progenitor cells in the normal brain, and is replaced by the expression of proteins specific for neurons or glia in differentiated cells. Furthermore, nestin is expressed not only in nervous system organs, but also in other organs and tissues such as the retina (Kohno et al. 2006), muscle (Kachinsky et al. 1994; Sejersen and Lendahl 1993), skin (Medina et al. 2006), liver (Forte et al. 2006), pancreas (Ueno et al. 2005), the testes (Frojdman et al. 1997), and others. We found significantly greater amounts of this protein in glaucomatous AH than in controls (Izzotti et al. 2010a). It has been suggested that nestin is expressed in dividing cells during the early stages of development and on differentiation, is downregulated and replaced by tissue-specific IF proteins. Although it remains unclear what factors regulate in vitro and in vivo expression of nestin, this protein is significantly more widespread than was previously thought. Mature neurons in the adult brain express nestin in the lateral ventricle and the dentate gyrus zone (Hendrickson et al. 2011). Furthermore, the fact that nestin expression in the endothelial cells of adult tissues is replenished by angiogenesis and in the endothelium of vascular neoplasms (Shimizu et al. 2006) and cancers (Aihara et al. 2004; Teranishi et al. 2002) suggests that nestin is also a marker of angiogenesis (Suzuki et al. 2010). Indeed nestin is expressed in proliferating endothelial cells and may be useful as a marker protein for neovascularization (Suzuki et al. 2010). This protein also participates in the formation of the cytoskeleton of newly formed endothelial cells (Mokrý et al. 2004). Our study confirmed the presence of nestin in glaucomatous AH, probably due to a response of the TM endothelial cells to injury. In glaucoma a dysfunction of the trabecular meshwork endothelium occurs which manifests itself through a hypertrophy and proliferation at the initial stages (Fine et al. 1981; Kuleshova et al. 2008). In addition, nestin has been found to be closely related to the cell membrane, intercellular junctions, and the nuclear membrane (Djabali 1999; Fuchs and Yang 1999) and is released upon the occurrence of cell damage (Yang et al. 1997; Vaittinen et al. 2001). Accordingly, its presence in AH in normal subjects may be due to physiological damage to TM endothelial cells, as occurs in response to the presence of free radicals in healthy conditions (Izzotti et al. 2009).

It is interesting that in glaucoma, this protein is used later to activate the glia, becoming a precise molecular signal for the retina, optic nerve and central nervous system (Saccà et al. 2016a). Furthermore nestin may be involved in mechanisms such as cell migration, generation of new neurons or glial cells and/or in retinal remodeling (Valamanesh et al. 2013).

In this complex scenario, the presence of nestin is indicative of the disintegration of the trabecular meshwork and its function which is to regulate the outflow of AH from the AC to Schlemm’s Canal. Therefore, a hypertonus occurs which in some way also determines optical atrophy. However, the intraocular pressure increase may be the consequence of the dysfunction of the endothelial cells of the trabecular meshwork and the hypertonus its epiphenomenon. The change of the proteoma in glaucoma precisely describes this alteration. The interesting thing is that the same proteins that can be found in the aqueous humor can also be found in the posterior segment (Steely et al. 2000). In order to determine the degree of uveoscleral outflow in the pony, Smith et al. (1986) 1- and 3-microns (diam) microspheres were perfused through the anterior chamber for 60 and 90 min. After 90 min, 1- and 3-microns spheres penetrated the prominent supraciliary space and mixed with the suprachoroidea of the midchoroid. The 1-micron spheres infiltrated tissues more extensively than did the 3-micron spheres, packing into the anterior meshwork and supraciliary space and also moving as far posterior as the suprachoroidea of the peripapillary retina. Therefore, it is more than likely that these proteins may also end up in the posterior segment, if so, it is probable that AKAP 2 plays critical roles in the control of excitability neurons and K⁺ transport. Synaptic plasticity is the ability of the connection between two neurons to change in strength in response to either the use or disuse of transmission over synaptic pathways. Synaptic plasticity plays important roles during normal postnatal development in learning and memory and in neurodegenerative diseases such as Alzheimer’s. The increase or decrease in synaptic plasticity, and hence in the strength of synaptic transmission, occurs through postsynaptic AMPA-type glutamate receptors. Both actions are induced by activation of NMDA-type glutamate receptors but differ in the level and duration of Ca²⁺ influx through the NMDA receptor and the subsequent engagement of downstream signaling by protein kinases including PKA, PKC and others (Sanderson and Dell’Acqua 2011). Moreover, AKAP controls NMDA and AMPA receptor function and hence synaptic plasticity through its interaction with two Ca²⁺-binding proteins caldendrin and calmodulin (Sekiguchi et al. 2013). Glutamate is a central nervous system excitatory neurotransmitter and has a central role in the conduction of signals between neurons. However, high extracellular levels of glutamate can induce neuronal cell death by excitotoxicity (Choi 1988). This phenomenon is well known at the level of the retina, and is determined by excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death (Lucas and Newhouse 1957). The ionotropic glutamate receptors have been classified into three major subtypes, AMPA, kainate, and N-methyl-D-aspartate (NMDA) receptors, named after their most selective agonist (Watkins et al. 1981). AMPA receptors are responsible for primary depolarization in glutamate-mediated neurotransmission and play key roles in synaptic plasticity. In glaucoma, the role of glutamate excitotoxicity remains unclear, not least because RGCs are relatively resistant to glutamate excitotoxicity in the presence of neurotrophic factors (Ullian et al. 2004).

The most abundantly expressed proteins in POAG explain both TM impairment and how these proteins can in turn be used as signals that spread in the posterior segment in the peri-papillary area. It is therefore conceivable that nestin, which is expressed in the anterior segment to activate stem cells present in the trabecular meshwork (Sacca et al. 2016b), functions in the posterior segment as an activator for glia. By contrast, AKAP 2 which in the posterior segment testifies to the breakdown of TM motility, in the posterior segment could be a signal for the apoptosis of retinal ganglion cells. Figure 14.6 summarizes the molecular events that lead to apoptosis.

Fig. 14.6

Diagram of the molecular events that lead to the decay of the trabecular meshwork, determining apotosis both in the anterior segment and at the level of the optic nerve head

Apoptosis of Retinal Ganglion Cells in Glaucoma

Apoptosis is the prevalent cause of neuronal cells death in neurodegenerative diseases (Honig and Rosenberg 2000). It is a highly regulated and controlled process characterized by biochemical events that lead to characteristic changes in cell morphology and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decrease (Elmore 2007).

Retinal ganglion cells (RCGs) have been demonstrated to die by apoptosis in several models of experimental optic nerve lesions and in human glaucoma, and Cordeiro et al. (2004) showed in vivo apoptotic RCGs death in experimental ocular hypertension in rats. The processes that lead to apoptosis during glaucoma include various stimuli (i.e. mitochondrial dysfunction, inflammation, ischemia, neurotoxicity, neurotrophic factor deprivation, increased intracellular calcium concentration) and this process generally involves two pathways: the intrinsic and extrinsic pathways (Almasieh et al. 2012; Nickells 1999). The intrinsic pathway, also called the mitochondrial pathway, is modulated by intracellular signals produced when cells are stressed and is based on the release of specific proteins from the intermembrane space of mitochondria. Conversely, the extrinsic pathway is modulated by extracellular molecules which bind to cell-surface death receptors, and leads to the production of the death-inducing signaling complex (DISC) (Elmore 2007).

Intrinsic Pathway

Mitochondria are well-known coordination center for apoptosis. Indeed, many pro- or anti-apoptotic signals converge on the mitochondria (Izzotti et al. 2010a, b). When there is an imbalance in mitochondria, owing apoptosis, we observe an increase in the permeability of the mitochondrial membrane and the release of a variety of mediators of apoptosis (Scaffidi et al. 1998). One of the most important molecules involved in apoptosis is cytochrome c which binds in the cytoplasm the apoptotic protease-activating factor-1 (Apaf-1) to form the apoptosome, thereby initiating apoptosis (Qu et al. 2010). It has been shown that experimental optic nerve damage cause early cytochrome c release in RGCs (Cheung et al. 2003). However, in a chronic disease such as glaucoma, the persisting mitochondrial dysfunction is believed to have an important role in the RCG loss. Of note, mitochondrial dysfunction is widely accepted to be one of the most important features of many neurological diseases such as Alzheimer’s disease, Parkinson’s disease and, glaucoma (Kong et al. 2009; Kumar 2016). Healthy RCG cells have a high metabolic rate and their soma contains a large number of mitochondria, in order to support the elevated demand for energy. In glaucoma, mitochondrial DNA has been found to be damaged; this could reflects a decrease in the respiratory activity and energy production for the cell resulting in dysfunction and possibly apoptosis (Almasieh et al. 2012). Interestingly, a negative correlation has been observed between age and both ATP levels in the optic nerve and intraocular pressure in mice (Baltan et al. 2010).

Moreover, damage to the mitochondria of RGCs may generate high levels of ROS causing the oxidation of lipids, proteins and mitochondrial DNA and creating a vicious circle that leads to their further deterioration (Izzotti et al. 2010).

The mitogen-activated protein kinases (MAPKs) are a broad family of protein Ser/Thr kinases that convert extracellular signals to series of molecular events, which ultimately result in a cellular response, and are central regulators of many cellular functions (Cargnello and Roux 2011). Among the MAPKs, a subfamily called Janus kinases (JNKs) has been demonstrated to be up-regulated in RGGs in animal models and in human glaucoma (Tezel et al. 2003). Moreover, administration of Janus kinase inhibitors has proved to be protective against RCG apoptosis in experimental ocular hypertension (Sun et al. 2011). Another subfamily of the MAPKs is that of the p38 MAPKs; these are over-expressed in glaucoma and their inhibition has proved to reduce the apoptosis of RGCs (Kikuchi et al. 2000). The fact that these MAPKs have been found to be associated with glaucomatous damage, does not prove their etiologic role. However, they may constitute a target for novel glaucoma therapies.

Bcl-2 family members are another class of proteins involved in the apoptotic process of the RGCs. Among them, the Bcl-XL is the predominant anti-apoptotic protein in the rat retina, and Bax also plays an important role in apoptosis (Elmore 2007). However, there is a lack of evidence that modulation of Bax expression by means of small interfering RNA (siRNA) promotes RCGs survival in animal models (Lingor et al. 2005). Again, identifying novel actors of the RCGs apoptotic pathway may open the door in future to innovative glaucoma therapies.

Extrinsic Pathway

TNF-alpha is the cytokine that has the most important role in the extrinsic pathway of apoptosis. When TNF-alpha binds to TNF-alpha receptors (TNFR1, TNFR2), it is able to initiate a cascade that leads to caspase activation and cell apoptosis (Elmore 2007). Of note, elevated values of TNF-alpha have been found in the brain, cerebrospinal fluid, and serum of patients affected by neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and, multiple sclerosis (Sriram and O’Callaghan 2007). Indeed, intravitreal injection of TNF-alpha is associated with RGC loss and TNF-alpha levels in optic nerve axons and in the aqueous humor of glaucoma patients level have been seen to increase (Tezel 2008). Although inhibition of TNF-alpha activity in the retina has been seen to result in marked RGC neuroprotection and TNF-alpha inhibitors are currently approved for the treatment of several autoimmune diseases such as rheumatoid arthritis and psoriasis, the role of such inhibitors in the treatment of glaucoma has not been yet demonstrated.

Triggers of RCG Apoptosis

In experimental glaucoma, a model of trans-synaptic degeneration has been described (Gupta and Yücel 2001), whereby damage spreads through synaptic connections, gradually influencing the whole neural pathway, from the retro-laminar portion of the ON to the distal portion at the level of the orbital apex (Bolacchi et al. 2012). Axonal transport is critical to the correct functioning of neurons, while the retrograde transport of neurotrophins may be necessary for the survival of RGCs. One of these neurotrophins is brain-derived neurotrophic factor (BDNF). BDNF is essential for the growth and survival of nerve cells (Sampaio et al. 2017). Conversely, excitotoxicity may also be associated with apoptosis of RGCs (Morrone et al. 2015). Excitotoxicity is the pathological process that leads to the death of neurons exposed to over-activation of the N-methyl-D-aspartate (NMDA) glutamate receptors (Prentice et al. 2015). Excessive activation of the NMDA receptor causes an influx of ions into the cell, in particular Ca²⁺. The excessive influx of calcium activates enzymes that degrade the cell membranes, cellular proteins, and nucleic acids, and eventually leads to apoptosis (Saccà and Izzotti 2008). It has been observed that the level of glutamate in the vitreous is increased in glaucoma patients and animal models of glaucoma. These data may suggest the involvement of excitotoxicity in RGC apoptosis (Dreyer and Grosskreutz 1997).

Another recently recent hypothesis (Saccà et al. 2016) speculates that pro-apoptotic proteins could pass from the anterior chamber to the posterior segment, becoming biological signals for the retina. Indeed, there is a pathway through which molecules pass from the anterior chamber to the optic nerve head, through the suprachoroidal space (Smith et al. 1986). Hence, these proteins may reach the retina, and in particular the peripapillary area. Indeed, while the nestin expressed in the AC by TM cells attracts stem cells in an attempt to repair the TM, in the retina it could activate glia (Wang et al. 2000; Xue et al. 2006).The failure of optic nerve glia to clear axonal debris may lead to the accumulation of toxic proteins and hence to neurodegeneration. Therefore, the proteins secreted by the damaged TM endothelial cells might become molecular messengers that enable communication between AC structures and the ONH, thereby playing a role in the RGC apoptosis that characterizes glaucomatous optic neuropathy. Translational research is in progress to further elucidate this pathogenic mechanism.

Finally, we must also briefly mention the role that autophagy plays in cell death. Autophagy is a cellular process of lysosomal degradation that is essential for survival, differentiation, development and homeostasis. The relationship between ROS formation and mitochondrial metabolism is essential to cell homeostasis, and therefore also to mitochondria, in that it regulates immune responses and autophagy (Dan Dunn et al. 2015). Mitochondrial dysfunction, as we have seen, plays a fundamental role in neurodegeneration and glaucoma. Damaged mitochondria generate further ROS, especially if mitophagy is insufficient (Pryde et al. 2016). Mitophagy is a process of selective autophagic destruction of mitochondria that have become defective as a result of stress or damage, through autophagy. It is a process of selective destruction of defective mitochondria as a result of stress or damage. Autophagy also takes place constitutively in RGCs. Indeed, acute IOP elevation induces a reduction in markers of autophagy, suggesting a possible role of IOP in disrupting the retinal autophagic mechanism. This supports the notion that autophagy exerts a neuroprotective effect in the retina, and suggests that autophagy dysfunction may have a key role in the neuronal degeneration processes occurring in both glaucoma and Alzheimer disease (Nucci et al. 2013). This dysfunction is influenced by myocilin. The turnover of endogenous myocilin involves the ubiquitin-proteasome and lysosomal pathways. When myocilin is up-regulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced (Qiu et al. 2014). Induced autophagy has also been demonstrated in vivo in retinal ganglion cells of transgenic mice with optineurin mutation (Shen et al. 2011). Finally, numerous studies have indicated that autophagy declines with age, and that its induction can promote longevity (Madeo et al. 2010; Morselli et al. 2010). The relevance of the autophagic pathway in the context of glaucoma-associated RGC death and how this contributes to the pathology is still unclear. Furthermore, a number of studies have focused on autophagy as a potential target for pharmacological modulation in order to achieve neuroprotection (Russo et al. 2015).

Conclusions

Some changes in the visual field are age-related rather than disease-related. Indeed, it is known that after the age of 40 years the peripheral visual field deteriorates, probably owing to a progressive decay of the peripheral lamina choriocapillaris (Rutkowski and May 2017). It remains to be ascertained whether these defects have a real impact on the evaluation of the possible presence of disease. VF is the only direct method which allows us to measure the visual function of patients and, therefore, also in the case of glaucoma; it also enables to make a point-to-point map of the retinal function projected in space (Fig. 14.7). Therefore, it allows us to assess disease progression and consequently, the effectiveness of treatment to slow down this progression and the loss of VF. Fortunately, progression is very fast only in 6–13% of cases (Chen 2003). Heijl et al. (2012) have calculated that in only 15% is a progression of the VF defect greater than −1.5 dB/year. Glaucoma damage primarily affects the optic nerve fibers entering the upper and lower poles and coming from the ganglionic temporal retina cells. Extending along the papillomacular beam in an arch-like fashion and respecting the horizontal meridian. The initial damage occurs as a paracentral scotoma prevalently nasal to the blind spot. Automatic perimetry is now the standard examination. Unfortunately, however, the great individual variability of the VF determines the initial examination (Heijl et al. 1989) (not everyone has the skill to do this test well!); consequently, frequent monitoring and/or a long period of time is required in order to accurately detect glaucomatous progression (Gardiner and Crabb 2002; Chauhan et al. 2008). Furthermore, the subjects have no symptoms except in the advanced stages of the disease. Thus, little is known about the precise relationship between functional measurements made in the clinic and the patient’s visual disability.

Fig. 14.7

Glaucomatous optic nerve (right eye) (a) Dense glaucomatous arcuate scotoma. (b) Glaucomatous optic disc with evident narrowing of the superotemporal and inferotemporal neuroretinal rim. (c) Box 1: Probability map of the ganglion cell layer (GCL) of the retina. Each pixel in the box is color-coded: no color (within the normal limit), yellow (outside 95% of the normal limit), or red (outside 99% of the normal limit). Box 2 shows the retinal nerve fiber layer (RNFL). The superior arcuate defect of the GCL and RNFL correlates with the inferior defect shown in the visual field (a)

Some VF defects, even advanced VF loss do not compromise the normal quality of life (Glen et al. 2012; Burton et al. 2014); in other cases, however quality of life is seriously impaired even if the disease is mild or moderate (Alqudah et al. 2016). In any case, vision-related quality of life first declines gradually, and then more quickly when functional abilities are significantly compromised (Jones et al. 2017). It is probable that the degree of quality of life impairment is in strictly dependent once the site of the VF damage (Fig. 14.8). Indeed, a loss of peripheral vision can compromise a person’s ability to move around safety in the environment. By contrast, a loss of central vision is a very serious obstacle to visual performance. Indeed, progressive loss of binocular RNFL thickness is associated with longitudinal loss of quality of life, even after adjustment for progressive visual field loss (Gracitelli et al. 2015). The loss of axons that occurs with ageing (Calkins 2013) is about 40% over the lifespan (Neufeld and Gachie 2003); photoreceptors also show a decline, 30% over a lifetime, and while the number of cones remains stable (Curcio et al. 1993), this natural evolution does not affect VF in healthy subjects. This visual decline is probably caused by diminished mitochondrial efficiency (Toescu 2005) with the result that the homeostatic balance of intracellular Ca²⁺ is lost, Na⁺/K⁺ -ATPase activity decreases, and increased oxidative injury occurs (Toescu and Verkhratsky 2000). From a clinical point of view the visual disability of glaucoma patients can be quantified in a suitable environment by measuring the patient’s actual performance of real-life tasks (Wei et al. 2012; Crabb 2016). Indeed, it is now known that many patients with advanced visual field loss, even with preserved visual acuity, have measurable difficulty in reading (Burton et al. 2014). These subjects have difficulty in grasping objects (Kotecha et al. 2009), (Fig. 14.9), and those with a scotoma in the inferior VF are more likely to fall (Kotecha et al. 2012). In addition, these subjects have reduced ability to recognize obstacles while driving (Haymes et al. 2008; Blane 2016), and have difficulty locating everyday objects and recognizing faces (Smith et al. 2011; Glen et al. 2012). Therefore, how do patients who have a VF defect see? Certainly the damaged portion of the VF cannot be replaced by undamaged portions. However, the brain has the ability to complete and to reconstruct the missing VF portions. This means that patients suffering from abnormal VF may not perceive their impaired vision as a problem or a disability (Crabb 2016). However, as in all patients with neurodegenerative diseases, the problem that exists is real and can actually be dangerous for themselves and for others.

Fig. 14.8

Examples of different visual field defects that can occur in different phases of neurodegenerative diseases or in glaucoma. These defects may proceed differently in the upper and lower portions of the VF, though the upper VF is more frequently involved in the early stages of glaucoma

Fig. 14.9

A person with visual impairment due to neurodegeneration is likely to have vision in which the brain is able to reconstruct the picture seen. However, the details are blurred owing to the VF damage (a) compared with normal vision (b).Tunnel vision does not adequately describe the visual experience of patients: indeed, it is very tough for a patient to describe his visual defect in terms of “black” or “tunnel”, the terms usually used in books to describe these types of visual defects. Affected patients usually use words such as “foggy”, “fuzzy”, or “unfocused” (Crabb et al. 2013). In this figure, the patient’s has a visual defect affecting his upper right side (a)