**3. Retinal degeneration in AD transgenic mice**

cytoskeletal assembly in neurons. Through the phosphorylation of tau, microtubule assembly

**Figure 1.** Schematic diagram summarizing the roles of tau in retinal functions. Tau stabilizes microtubules. Dislocation of Tau from microtubules can result in growth cone collapse. Accumulation of β-amyloid (Aβ) is an example to trigger phosphorylation of tau and hence detaches from microtubule. Apart from Aβ as a triggering factor, any stimulation of signaling cascade of cdk5, ephrin-A receptor or calmodulin-dependent protein kinase II affecting phosphorylation of tau can also modulate microtubules. Once tau leaves microtubules after phosphorylation, they can easily form aggre‐ gation, which can further impair axonal transport mediated by kinesin or dynein. Consequently, mitochondria in the distal part of nerve, nerve terminus or spines cannot obtain protection from the cell body (soma) so that they are col‐

**Figure 2. Chiu et al., 2012**

proteins Growth cone collapse

ephrin-As Axonal growth regulation

Survival factors and oxidative protector transport decreased

Cytoskeleton modification

Abnormally aggregated tau inhibits the transportation of mitochondria by kinesin-like motors towards the cell periphery of rat RGCs. Consequently, neurons with perturbation of mito‐ chondria and peroxisomes suffer from loss of energy production and accumulation of reactive oxygen species (ROS). The anterograde transport of vesicles required for growth cones and synaptic function is retarded. In addition, these neurons may be more vulnerable to oxidative stress[40]. In the RGCs axons of P301S mutant mice, the projection domain of tau interacts with the C-terminus of p150, the major component of the dynein-activator. The co-localization of tau and p150 suggests that tau dysfunction can result in the mislocalization of dynactin in

**2. Clinical manifestation of visual deficits in Alzheimer's patients**

Various visual deficits in AD have been reported since 1987 [42]. Cognitive visual changes have been reported in patients in the early stages of AD [43], including difficulties in reading

may be inhibited; and hence, the cellular architecture is disrupted[39].

lapse and cannot produce energy. Neurodegeneration can unavoidably occur.

Transport proteins E.g. dynein, kinesin,

P150

Cytoskeletal

Cdk5/p35 and

Calmodulin kinase II system

Beta amyloid accumulation

axons, which can result in neurodegeneration[41].

**Tau**

160 Glaucoma - Basic and Clinical Aspects

Since there is a lack of postmortem human retinal samples from AD patients, progress of investigating pathogenesis of retinal neurodegeneration in the AD eyes is slow. Much of the insights have gained from specific gene mutations that account for the familial AD (FAD). The majority (30-40%) of FAD is resulted from autosomal dominant inheritance with mutated genes encoding presenilin 1 (PS1) on the long arm of chromosome 14, presenilin 2 (PS2) on chromosome 1 and amyloid precursor protein (APP) on chromosome 21 [68]. Depending on the number of genes they express, transgenic mice come in three varieties: single (APP, PS1, or Tau), double (APP/PS1, APP/Tau), or triple transgenic (APP/PS1/ Tau) [69]. Behavioral studies on AD transgenic mice showed that the mice were suffered from visual dysfunction [70].

**3.3. Deposition of Aβ in the retina and retinal vasculature**

**Neuronal cell loss^**

Yes +++

n/a +++

n/a ++

Yes +++

Yes +

Yes +++

**Table 1.** Retinal changes in documented AD transgenic mouse models

**APP overexpression**

(GCL, INL)

(GCL, INL)

(IPL, OPL)

(GCL, INL)

(NFL,GCL, IPL, INL, OPL, OS, RPE, RV)

Yes n/a \*Yes

(GCL)

± (GCL, RV)

±

± (NFL,CV)

+++ (NFL, GCL) + (RV, CV)

n/a: not applicable; *swe*: Swedish mutation; *P-tau*: hyperphosphorylated tau; ^ neuronal cells in the inner retinal regions, including INL and GCL; \* plaques formation; *NFL*: nerve fiber layer; *GCL*: ganglion cell layer; *IPL*: inner plexiform layer; *INL*: inner nuclear layer; *OPL*: outer plexiform layer; *ONL*: outer nuclear layer; *OS*: outer segment; *RPE*: retinal pigment epithelium; *RV*: retinal vasculature; *CV*: choroidal vasculature; +++ strong level; ++ moderate level; + weak level; +/-

(GCL, INL, RV)

No n/a \*(IPL, OPL) n/a n/a [77]

No n/a n/a n/a ±(IPL), with

(layers not specified)

expressing APP/PS1.

**Mutant genes Type/**

APPswe, PS1∆E9 Double/

APPswe, PS1∆E9 Double/

APPswe, PS1∆E9 Double/

APPswe, PS1M146L Double/

APPswe, PS1M146L Double/

TauP301S Single/

PS1M146V, APPswe, and TauP301L

present.

APPswe, (HuAPP695. K670N, M671L)

APPswe, (HuAPP695. K595N, M596L) **Age**

Single/ 14 months

Single/ 2-to18-months

6-to 12-months

10.5-months

7.8 months

27-months

1- to 6-months

Triple/ 10-to 22 months

12-to 19-months

The deposition of Aß, derived from abnormal processing of APP was found in the retinas of AD transgenic mice. In single transgenic Tg2576 mice, Aβ was found to deposit from RGCL to INL or even at ONL. Aβ deposits and plaque like formation were detected by four different monoclonal antibodies such as BAM01, 6E10, Aβ-40 and Aβ-42 as well as Congo red stain‐ ing[73], [76]. Retinal Aβ deposit has also been found in double transgenic mouse models

**Aβ deposits Aβ-deposited**

Progressive Neurodegeneration of Retina in Alzheimer's Disease — Are β-Amyloid Peptide…

+++ (GCL, IPL, INL, OPL, ONL, RV) + (OS)

**vasculature**

Yes ±

**P-tau deposits**

(GCL, IPL, INL, OPL, ONL)

Yes n/a [75]

Yes n/a [75]

Yes n/a [11]

Yes n/a [11]

Yes n/a [11]

paired helical filament formation

n/a n/a [79]

[78]

**Ref.**

http://dx.doi.org/10.5772/53428

163

[73]

Multiple lines of AD transgenic mice have elicited AD-like pathological hallmarks in the retina as disease progresses [69]. The over-expression of APP, the production of soluble Aβ, and Aβ deposition will lead to formation of amyloid plaques that can induce cell death via the apoptotic pathway [71]. Even before formation of plaques, oligomeric Aβ can induce synaptic degeneration. Furthermore, Aβ plays a role in inducing hyperphosphorylation of tau, which in turn affects the integrity of retinal cells and their synapses in inner nuclear layer (INL) [72]. It has been reported that over-expression of APP, Aβ and/or tau deposition, neuronal cell loss, changes of retinal glial cell, and vascular changes occur in the retina of AD transgenic mice. The changes of retinal histopathology documented in the AD transgenic mouse models are summarized in Table 1.

#### **3.1. Loss of retinal neurons in AD transgenic mice**

In consistent with the findings in the retina of AD patient, reduced retinal thickness between RGCL and ONL was detected in Tg2576 mice [73]. This indicates that there was a loss of either the photoreceptor cells in the ONL (rod and cone cells) or neuronal cells in the inner retinal layers (RGC, horizontal cells, bipolar cells or amacrine cells). In double transgenic mice strain (APPswe/PS1M146L), a significant increase in the number of apoptotic cells in the RGCL was detected by TUNEL staining as the animal grew from 7.8 months to 27 months [11]. By using a different double transgenic mice strain (APPswe/PS1∆E9), there was significant increase of TUNEL-positive cells in the RGCL when comparing with age-matched controls. Most recently, direct visualization of apoptotic RGCs in the retina was reported in triple transgenic mouse model of AD [74]. Using a fluorophore-labeled annexin V protein as a marker of apoptosis and cSLO to detect the fluorescence, the triple transgenic mice displayed increased number of RGC apoptosis compared with wild-type controls.

#### **3.2. Over-expression of APP in the AD retina**

Compared to the wild-type mice, a significant increase in immunoreactivity of APP in the cytoplasm was detected in RGCL and INL of various transgenic mice [11], [73], [74]. In single transgenic Tg2576 mice, over expressed APP was detected in the RGCL and INL of 14-monthold mice [73], [75]. In double transgenic mice strain APPswe/PS1M146L, over-expression of APP was age-dependent. In 27 months old mice, immunoreactivity of APP was detected not only in the different layers of retina such as NFL, RGCL, inner plexiform layer (IPL), INL, outer plexiform layer (OPL), outer segment (OS) and retinal pigment epithelium (RPE) but also in the retinal vasculature [11]. By using different double transgenic strain- APPswe/PS1∆E9, APP immunoreactivity was exhibited in the RGCL only at an intermediate age of 10.5 months. In earlier time point (9 month-old animals), moderate immunoreactivity of APP was detected only in the IPL and OPL not in the RGCL [11], [73], [76], [77].

#### **3.3. Deposition of Aβ in the retina and retinal vasculature**

Depending on the number of genes they express, transgenic mice come in three varieties: single (APP, PS1, or Tau), double (APP/PS1, APP/Tau), or triple transgenic (APP/PS1/ Tau) [69]. Behavioral studies on AD transgenic mice showed that the mice were suffered

Multiple lines of AD transgenic mice have elicited AD-like pathological hallmarks in the retina as disease progresses [69]. The over-expression of APP, the production of soluble Aβ, and Aβ deposition will lead to formation of amyloid plaques that can induce cell death via the apoptotic pathway [71]. Even before formation of plaques, oligomeric Aβ can induce synaptic degeneration. Furthermore, Aβ plays a role in inducing hyperphosphorylation of tau, which in turn affects the integrity of retinal cells and their synapses in inner nuclear layer (INL) [72]. It has been reported that over-expression of APP, Aβ and/or tau deposition, neuronal cell loss, changes of retinal glial cell, and vascular changes occur in the retina of AD transgenic mice. The changes of retinal histopathology documented in the AD transgenic mouse models are

In consistent with the findings in the retina of AD patient, reduced retinal thickness between RGCL and ONL was detected in Tg2576 mice [73]. This indicates that there was a loss of either the photoreceptor cells in the ONL (rod and cone cells) or neuronal cells in the inner retinal layers (RGC, horizontal cells, bipolar cells or amacrine cells). In double transgenic mice strain (APPswe/PS1M146L), a significant increase in the number of apoptotic cells in the RGCL was detected by TUNEL staining as the animal grew from 7.8 months to 27 months [11]. By using a different double transgenic mice strain (APPswe/PS1∆E9), there was significant increase of TUNEL-positive cells in the RGCL when comparing with age-matched controls. Most recently, direct visualization of apoptotic RGCs in the retina was reported in triple transgenic mouse model of AD [74]. Using a fluorophore-labeled annexin V protein as a marker of apoptosis and cSLO to detect the fluorescence, the triple transgenic mice displayed increased number of RGC

Compared to the wild-type mice, a significant increase in immunoreactivity of APP in the cytoplasm was detected in RGCL and INL of various transgenic mice [11], [73], [74]. In single transgenic Tg2576 mice, over expressed APP was detected in the RGCL and INL of 14-monthold mice [73], [75]. In double transgenic mice strain APPswe/PS1M146L, over-expression of APP was age-dependent. In 27 months old mice, immunoreactivity of APP was detected not only in the different layers of retina such as NFL, RGCL, inner plexiform layer (IPL), INL, outer plexiform layer (OPL), outer segment (OS) and retinal pigment epithelium (RPE) but also in the retinal vasculature [11]. By using different double transgenic strain- APPswe/PS1∆E9, APP immunoreactivity was exhibited in the RGCL only at an intermediate age of 10.5 months. In earlier time point (9 month-old animals), moderate immunoreactivity of APP was detected

from visual dysfunction [70].

162 Glaucoma - Basic and Clinical Aspects

summarized in Table 1.

**3.1. Loss of retinal neurons in AD transgenic mice**

apoptosis compared with wild-type controls.

**3.2. Over-expression of APP in the AD retina**

only in the IPL and OPL not in the RGCL [11], [73], [76], [77].

The deposition of Aß, derived from abnormal processing of APP was found in the retinas of AD transgenic mice. In single transgenic Tg2576 mice, Aβ was found to deposit from RGCL to INL or even at ONL. Aβ deposits and plaque like formation were detected by four different monoclonal antibodies such as BAM01, 6E10, Aβ-40 and Aβ-42 as well as Congo red stain‐ ing[73], [76]. Retinal Aβ deposit has also been found in double transgenic mouse models expressing APP/PS1.


n/a: not applicable; *swe*: Swedish mutation; *P-tau*: hyperphosphorylated tau; ^ neuronal cells in the inner retinal regions, including INL and GCL; \* plaques formation; *NFL*: nerve fiber layer; *GCL*: ganglion cell layer; *IPL*: inner plexiform layer; *INL*: inner nuclear layer; *OPL*: outer plexiform layer; *ONL*: outer nuclear layer; *OS*: outer segment; *RPE*: retinal pigment epithelium; *RV*: retinal vasculature; *CV*: choroidal vasculature; +++ strong level; ++ moderate level; + weak level; +/ present.

**Table 1.** Retinal changes in documented AD transgenic mouse models

In double transgenic mice strain APPswe/PS1M146L, Aβ was found to deposit predominantly in NFL and RGCL in aged mice of 27 months but not at young mice of 7.8 months. In another double transgenic strain APPswe/PS1∆E9, similar Aβ deposition was detected in intermediate age of 10.5 months[11]. In another subsequent study using transgenic mice APPswe/PS1∆E9, Aβ plaques were found by thioflavin-S staining in plexiform layers; the size and the number of plaques significantly increased with age from 12 months[77]. The transparent nature of the eyes allows direct tracking and visualization of the Aβ signal has also been detected in the retinal and choroidal vasculature. In single transgenic Tg2576 mice, Aβ was detected around microvessels in RGCL[73], [76]. Both retinal and choroidal vascular Aβ deposits were reported in aged (27 months) APPswe/PS1M146L transgenic mice and intermediate-aged (10.5 months) APPswe/PS1∆E9 mice [11].

RGC detected by using Aβ antibody was co-localized with apoptotic RGC cells. Targeting Aβ pathway in this experimental model, three different approaches were applied including: (*i*) β-secretase inhibitor to reduce formation of Aβ; (*ii*) an anti-Aβ antibody to clear Aβ deposition; and (*iii*) Congo red to inhibit aggregation of Aβ and neurotoxic effects of Aß. Manipulating production of Aβ pathway, apoptosis of RGC was successfully reduced by suppressing further Aβ aggregation and inhibiting the enzymatic activity of amyloidogenesis. The combined treatment (triple therapy) was more effective than either single- or dual-agent therapy in protecting RGC survival under COH. Increased expression of Aβ in the RGCL and optic nerve was related to abnormal APP-splicing in the presence of elevated IOP in DBA/2J glaucomatous mouse retinas [82] and mouse experimental COH model [83]. Increase of Aβ in the retinal in COH has been found to be associated with activation of caspase-8 and caspase-3, and caspase-3-mediated APP cleavage product (DeltaC-APP) in the RGCs under COH [65]. Application of exogenous Aβ peptide into the vitreous also induced significant RGC apoptosis

Progressive Neurodegeneration of Retina in Alzheimer's Disease — Are β-Amyloid Peptide…

http://dx.doi.org/10.5772/53428

165

There are some suggestions that Aβ peptides modulate Ca2+ level in mitochondria that may alter the mitochondrial morphology and physiology [84]. For examples, elevated cytosolic Ca2+ levels may enhance fragmentation of mitochondria. This can lead to the perturbation of fission and fusion balance, which may eventually cause mitochondrial dysfunction [85]. Dysregulation of Ca2+ homeostasis may also disrupt downstream pathways of Ca2+-dependent regulators monitoring mitochondrial dynamics [84], [86]. Consequently, synaptic dysfunction may occur due to the failure of meeting the energy demand in neurons, particularly in axonal

Our eyes are energy demanding organs in which high density of mitochondria exist at the optic nerve heads [89]. If one applies Aβ to the eyes, it may trigger mitochondrial dysfunctions resulting in retinal degeneration. Intriguingly, in a glaucomatous model where cultured RGCs were subjected to elevated hydrostatic pressure, fission of mitochondria was found to be enhanced, together with morphological changes and bioenergetics dysfunction [90]. A clinical study showed that mean mitochondrial respiratory activity was decreased by 21% in patients with primary open-angle glaucoma compared with age-matched control subjects (p < 0.001) [91]. In rabbit model of COH, daily tropical application of 5 µM mitochondria targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) showed reduction in glaucomatous changes [92]. This hypothesis may be extended to one of the causes

In aged retina (49-87 year-old human), there is a positive correlation between age and number of tau-positive RGCs. Diffuse immunoreactivity of tau was found in the INL, while aggregated

in rat retina [81].

and dendritic terminals [86]-[88].

in Aβ-induced RGC apoptosis in glaucoma.

**5.1. Tau in the retina of glaucoma patient**

**5. TAU and glaucoma**

**4.2. Aβ-mediated mitochondrial dysfunction and glaucoma**

#### **3.4. Deposition of hyperphosphorylated tau in the retina**

Hyperphosphorylation of tau protein and subsequent deposition as neurofilbrillary tangles is associated with AD. Tau inclusions have been observed in the brains as well as in the retinas of Tg2576 and triple transgenic mice [79]. In single transgenic Tg2576 mice, hyperphosphory‐ lated tau was detected by antibody AT8 in various retinal layers from RGCL through to the ONL. The hyperphosphorylated tau was found to be associated with Aβ depositions [73]. Another single transgenic expresses human P301S tau transgene, hyperphosphorylated tau was found to deposit in the RNFL and aggregated into filamentous inclusions in RGCs starting from 2-month-old mice [78]. Hyperphosphorylation and aggregation of tau were associated *in vivo* with reduced axonal transport, both anterograde and retrograde, in the optic nerve of this transgenic mice line [80].

#### **3.5. Glial reaction in AD retina**

Glial reactions, activated microglia and astrocytes, in the retina were detected in different kinds of AD transgenic mice at various ages. In Tg2576 transgenic mice, significant infiltration of microglial cells detected by iba-1 and the increased astrocytes activation detected by GFAP in the inner retina were detected as early as 4-month-old mice [73]. In double transgenic APPswe/ PS1M146L mice, microglia was increased in an age-dependent manner, which was in parallel with Aß deposits and TUNEL positive RGC in the GCL. The average percentage of cells in the GCL surrounded by microglial cells increased significantly from 10% in 7.8-month-old to 50% in 27-month-old APPswe/PS1M146L transgenic mice [11]. In another double transgenic APPswe/ PS1∆E9 mice, qualitative evaluation revealed greater immunoreactivity of microglia in 12 to 19 months old transgenic mice when compared to age-matched non-transgenic control[77].

### **4. β-Amyloid peptide and glaucoma**

#### **4.1. Aβ in animal models mimic glaucoma**

In a rat model mimicking chronic ocular hypertension (COH) [81], Aβ has been reported to be implicated in the development of RGC apoptosis in glaucoma. Increased intracellular Aβ in RGC detected by using Aβ antibody was co-localized with apoptotic RGC cells. Targeting Aβ pathway in this experimental model, three different approaches were applied including: (*i*) β-secretase inhibitor to reduce formation of Aβ; (*ii*) an anti-Aβ antibody to clear Aβ deposition; and (*iii*) Congo red to inhibit aggregation of Aβ and neurotoxic effects of Aß. Manipulating production of Aβ pathway, apoptosis of RGC was successfully reduced by suppressing further Aβ aggregation and inhibiting the enzymatic activity of amyloidogenesis. The combined treatment (triple therapy) was more effective than either single- or dual-agent therapy in protecting RGC survival under COH. Increased expression of Aβ in the RGCL and optic nerve was related to abnormal APP-splicing in the presence of elevated IOP in DBA/2J glaucomatous mouse retinas [82] and mouse experimental COH model [83]. Increase of Aβ in the retinal in COH has been found to be associated with activation of caspase-8 and caspase-3, and caspase-3-mediated APP cleavage product (DeltaC-APP) in the RGCs under COH [65]. Application of exogenous Aβ peptide into the vitreous also induced significant RGC apoptosis in rat retina [81].

#### **4.2. Aβ-mediated mitochondrial dysfunction and glaucoma**

There are some suggestions that Aβ peptides modulate Ca2+ level in mitochondria that may alter the mitochondrial morphology and physiology [84]. For examples, elevated cytosolic Ca2+ levels may enhance fragmentation of mitochondria. This can lead to the perturbation of fission and fusion balance, which may eventually cause mitochondrial dysfunction [85]. Dysregulation of Ca2+ homeostasis may also disrupt downstream pathways of Ca2+-dependent regulators monitoring mitochondrial dynamics [84], [86]. Consequently, synaptic dysfunction may occur due to the failure of meeting the energy demand in neurons, particularly in axonal and dendritic terminals [86]-[88].

Our eyes are energy demanding organs in which high density of mitochondria exist at the optic nerve heads [89]. If one applies Aβ to the eyes, it may trigger mitochondrial dysfunctions resulting in retinal degeneration. Intriguingly, in a glaucomatous model where cultured RGCs were subjected to elevated hydrostatic pressure, fission of mitochondria was found to be enhanced, together with morphological changes and bioenergetics dysfunction [90]. A clinical study showed that mean mitochondrial respiratory activity was decreased by 21% in patients with primary open-angle glaucoma compared with age-matched control subjects (p < 0.001) [91]. In rabbit model of COH, daily tropical application of 5 µM mitochondria targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) showed reduction in glaucomatous changes [92]. This hypothesis may be extended to one of the causes in Aβ-induced RGC apoptosis in glaucoma.
