Retinal changes in the Tg-SwDI mouse model of Alzheimer’s disease

Highlights

• Data from a transgenic Alzheimer’s disease (AD) model indicate that retinal changes parallel AD pathology in the brain.

• Tg-SwDI mouse model of AD exhibited differential gene regulation of acetylcholine receptor subunits in the retina.

• Tg-SwDI AD mice had age-dependent loss of retinal cholinergic cells as compared to non-transgenic (wild-type) mice.

• Transgenic AD mice displayed significantly more gliosis than wild-type mice in the retinal nerve fiber layer.

Abstract

Alzheimer’s disease (AD), a debilitating neurodegenerative illness, is characterized by neuronal cell loss, mental deficits, and abnormalities in several neurotransmitter and protein systems. AD is also associated with visual disturbances, but their causes remain unidentified. We hypothesize that the visual disturbances stem from retinal changes, particularly changes in the retinal cholinergic system, and that the etiology in the retina parallels the etiology in the rest of the brain. To test our hypothesis, quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were employed to assess changes in acetylcholine receptor (AChR) gene expression, number of retinal cells, and astrocytic gliosis in the Transgenic Swedish, Dutch and Iowa (Tg-SwDI) mouse model as compared to age-matched wild-type (WT). We observed that Tg-SwDI mice showed an initial upregulation of AChR gene expression early on (young adults and middle-aged adults), but a downregulation later on (old adults). Furthermore, transgenic animals displayed significant cell loss in the photoreceptor layer and inner retina of the young adult animals, as well as specific cholinergic cell loss, and increased astrocytic gliosis in the middle-aged adult and old adult groups. Our results suggest that the changes observed in AD cerebrum are also present in the retina and may be, at least in part, responsible for the visual deficits associated with the disease.

Introduction

Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that affects over 26 million people worldwide and the incidence is projected to quadruple by the year 2050 (Tsai et al., 2014). According to the Alzheimer’s Association, in 2015 there were 5.3 million Americans suffering from AD. It is characterized by circadian rhythm dysfunction and the development of multiple cognitive deficits, including memory loss, confusion, apraxia, aphasia and agnosia (American Psychiatric Association, 2013, La Morgia et al., 2015).

AD is marked by the accumulation of neurofibrillary tangles (aggregates of hyper-phosphorylated tau protein), deposition of amyloid beta (Aβ) plaques, gliosis, and substantial neuronal and synaptic loss (Fodero et al., 2004). The pathophysiology of AD is extremely intricate and involves several biochemical pathways. These include defective Aβ protein metabolism and abnormalities of several neurotransmitter systems, particularly the cholinergic and glutamatergic systems (Doraiswamy, 2002, Francis et al., 2012).

In addition to cognitive decline and cortical changes, AD is also characterized by visual dysfunction ranging from simple (e.g. color discrimination) to complex (e.g. object recognition), including deficits in motion perception, contrast sensitivity, stereopsis, temporal resolution, acuity, color, and lower critical flicker fusion threshold (Cronin-Golomb et al., 1995, Rizzo et al., 2000). In 1906, Alois Alzheimer was the first to report the occurrence of visual disturbances in one of his patients Auguste D (Maurer et al., 1997, Kusne et al., 2016).

Visual deficits have been reported in the early stages of the disease, even before AD diagnosis is clearly established (Cronin-Golomb et al., 1991, Uhlmann et al., 1991). The effects of AD on visual attention and other higher visual functions can negatively impact one’s quotidian activities such as reading, route finding, object localization and recognition (Rizzo et al., 2000). To date, the underlying causes of these visual dysfunctions and whether they stem from retinal or cortical abnormalities remain poorly understood (Tsai et al., 2014).

The excitatory neurotransmitter acetylcholine (ACh) plays a crucial role in myriad cognitive functions, including learning and memory; both of which are negatively impacted by AD. In the brain, ACh is released by cholinergic neurons and can bind to two different acetylcholine receptor (AChR) subtypes: nicotinics (nAChRs) and muscarinics (mAChRs), which are ionotropic and metabotropic receptors, respectively (Oddo and LaFerla, 2006). In early AD, there is impairment in hippocampus-based episodic memory that is improved through enhancement of cholinergic transmission (Hernandez et al., 2010).

In the retina, ACh is synthesized and released by starburst amacrine cells (Masland, 1980). Release of ACh is both tonic and light-evoked (Masland, 1980). AChRs are expressed by photoreceptor, bipolar, amacrine, displaced amacrine, horizontal and ganglion cells in several different species (Dmitrieva et al., 2007, Strang et al., 2007, Strang et al., 2010, Cimini et al., 2008, Smith et al., 2014). AChR activation has been shown to play a role in retinal development (Stacy et al., 2005, Sun et al., 2008, Ford and Feller, 2012) and affect ganglion cell responses (Schmidt et al., 1987, Kittila and Massey, 1997, Strang et al., 2005, Strang et al., 2007, Strang et al., 2010, Strang et al., 2015).

The main animal models of AD were designed to mimic the autosomal dominant mutations observed in hereditary early onset Alzheimer’s. These models express mutations in amyloid precursor protein (APP) and/or in the presenilin proteins (PSEN1 and PSEN2). All of the identified mutations that cause autosomal dominant AD directly alter the production of Aβ through APP processing. APP is a type I transmembrane protein with a large amino-terminal extracellular domain (Hall and Roberson, 2013). Aβ is a peptide that stems from the cleavage of APP by the enzymes β-secretase and ϒ-secretase, which is composed of presenilin and other components (De Strooper et al., 1998, Edbauer et al., 2003).

Male and female transgenic Swedish, Dutch and Iowa (Tg-SwDI) mice were used for this study. These mice express the human APP, isoform 770, with the Swedish APP K670N/M671L, Dutch E693Q, and Iowa D694N mutations driven by the mouse Thy1 promoter (Murrell et al., 1991). The Dutch and Iowa are missense mutations that occur on exon 17. In the Dutch APP E693Q mutation, glutamic acid (GAA) is replaced by glutamine (CAA) (Levy et al., 1990).

The Dutch mutation leads to cell death and loss of vessel wall integrity (Wisniewski et al., 1991), and is associated with severe Aβ deposition in cerebral vessels, hemorrhages, and diffuse plaques in brain parenchyma (Timmers et al., 1990). The Iowa APP D694N mutation is characterized by the substitution of aspartic acid (GAT) by asparagine (AAT) (Grabowski et al., 2001). The Dutch and Iowa mutations occur within Aβ and result in increased resistance to proteolysis (Hall and Roberson, 2013). The Swedish APP K670N/M671L is a double mutation at the β-secretase cleavage site (Hall and Roberson, 2013), on exon 16, in which lysine (AAG) and methionine (AAT) are replaced by asparagine (AAT) and leucine (CTG) (Mullan et al., 1992). This mutation results in increased Aβ40 and Aβ42 (the more toxic form) production (Hall and Roberson, 2013).

In the Tg-SwDI mice, Aβ accumulation in the cerebrum is extensive by 12 months (Van Vickle et al., 2008). These mice show impaired learning and memory in the Barnes maze task as early as 3 months of age (Xu et al., 2007). At 6 months of age, these mice start developing gliosis with a prominent increase in the number of glial fibrillary acidic protein (GFAP) positive astrocytes in several brain regions (Miao et al., 2005).

Little is known about the retinal cholinergic system in many of the AD animal models and whether they display retinal abnormalities. Because these retinal changes may parallel AD etiology in the brain and precede severe cognitive impairment, they may be instrumental in the early diagnosis of AD. Thus, in the current study we assessed whether the AD-related changes in the retina are analogous to the alterations reported in the rest of the brain and identified possible causes for visual dysfunction by quantifying AChR gene expression, cholinergic cell count, total retinal cell count and astrocytic gliosis in Tg-SwDI mice as compared to wild-type (WT). The Tg-SwDI mice showed a decrease in the number of retinal cells, gliosis and alterations in AChRs expression.