Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model
Introduction
Alzheimer's disease (AD) is a common and devastating age-dependent neurodegenerative condition. At present, a definite diagnosis of AD is determined after brain autopsy by detecting accumulation of the hallmark proteolytic products of amyloid precursor protein (APP), β-amyloid peptides (Aβ), which form extracellular aggregates termed Aβ plaques, and by the presence of intracellular neurofibrillary tangles (Hardy & Selkoe, 2002, Sisodia & Price, 1995). Aβ plaques are believed to contribute to disrupted cellular activities and communication in the brain, leading to neurotoxic inflammation and neuronal death (McGeer & McGeer, 2002, Wyss-Coray, 2006). Major efforts have been invested in developing tools to enable noninvasive detection of amyloid plaques (e.g., through the skull) in living AD patients and animal models (Hintersteiner et al., 2005, Klunk et al., 2004, Nakada et al., 2008, Ng et al., 2007). However, such noninvasive monitoring of Aβ plaques is still clinically challenging, and is of limited resolution and availability (Klunk et al., 2005, Lockhart et al., 2007, Toyama et al., 2005). Optical detection constitutes a powerful, high-resolution and specific tool for in vivo imaging (Fujimoto and Farkas, 2009), as demonstrated using multiphoton microscopy to detect Aβ plaques in the mouse brain via an invasive cranial window (Meyer-Luehmann et al., 2008).
An alternative noninvasive approach to visualize amyloid plaques in AD patients at high resolution could be a direct optical imaging of the retina, provided that Aβ plaques are formed in patients’ retinas and share properties with those in the brain. Choosing the retina as a target for visualization of brain disease also relies on it being a direct extension of the brain, having the potential to faithfully reflect AD brain's pathology. Retinal abnormalities in AD patients, some of which were detected at early stages, have been described in the past (Berisha et al., 2007, Blanks et al., 1996, Hinton et al., 1986, Katz & Rimmer, 1989, Sadun et al., 1987, Trick et al., 1989). These changes, however, mostly related to the nerve fiber layer (NFL) thickness reduction, seem to appear in additional pathologies of the eye and the brain including ocular hypertension, glaucoma, demyelinating optic neuritis, multiple sclerosis, and Parkinson's disease (Jindahra et al., 2010, Parisi, 2003). Detection of ganglion cell death in retinas of live AD mouse models has been recently documented (Cordeiro et al., 2010); however, this phenomenon is common to multiple neurodegenerative disorders including glaucoma and age-related macular degeneration. In addition, postmortem lenses from AD patients were reported to exhibit specific cytosolic nanometer-size Aβ aggregates and supranuclear cataracts in the peripheral lens (Goldstein et al., 2003). Encouraging results in APPSWE and Presenilin (PS) 1∆E9 transgenic mice carrying the human mutated genes causing an early-onset familial AD were recently reported: these mice were found to develop human Aβ deposits in their retinal tissues at advanced stages of the disease (Ning et al., 2008, Perez et al., 2009).
The existing reports emphasized the need for unequivocal identification of Aβ plaque hallmark pathology of Alzheimer's disease in AD patients, especially at an early stage, for noninvasive in vivo detection of retinal plaques, and demonstration of their response to therapeutic intervention. Here, we report the presence of Aβ plaques in retinas of postmortem eyes from AD patients, and moreover, in retinas from those suspected as early stage cases. Using the APPSWE/PS1∆E9 transgenic (AD-Tg) mice, we provide evidence for the formation of Aβ plaques in the retina prior to their manifestation in the brain. Further, in these mice we found that an immune-based therapy, using a weak agonist of a myelin-derived peptide loaded onto dendritic cells (Koronyo-Hamaoui et al., 2009), was effective in reducing Aβ plaques in the retinas to an extent similar to that observed in the brain. Importantly, we were able to demonstrate that systemic injection of curcumin (diferuloylmethane), a natural and safe fluorochrome that binds and labels Aβ plaques (Garcia-Alloza et al., 2006, Yang et al., 2005), into live AD mice allows high-resolution and specific noninvasive in vivo visualization of retinal Aβ plaques.
Section snippets
Mice
Double transgenic mice (females and males in equal numbers) harboring the chimeric mouse/human APP (APPSWE) and mutant human presenilin 1 (PS1∆E9) genes, and their aged-matched wt littermates, were purchased from the Jackson Laboratories (Bar Harbor, ME, strain #4462). Both APPSWE and PS1∆E9 mutations are associated with early-onset Alzheimer's disease, and their expression is directed to CNS neurons. The “humanized” APPSWE transgene allows the mice to produce and secrete a human Aβ peptide.
Curcumin binds to retinal Aβ plaques in a mouse model of Alzheimer's disease
We first assessed the potential of using curcumin for the development of an in vivo noninvasive method to detect retinal Aβ plaques. To this end, we first verified that curcumin could specifically ex vivo label Aβ plaques in the retinas of AD-Tg mice. Retinal whole-mounts and cross-sections from AD-Tg and non-Tg (wt) mice were co-stained with curcumin and four different anti-Aβ mAbs (Fig. 1). Retinal Aβ plaques, co-labeled with curcumin and 11A5-B10 or 12F4 mAbs [recognizing the C-terminal
Discussion
The present study demonstrates that a noninvasive in vivo monitoring of AD hallmark pathology via optical imaging with high specificity and resolution is feasible through the retina. Two key issues towards translation of this approach for the detection of Alzheimer's in humans have been accomplished here: in vivo imaging of Aβ plaques in the retina of live AD animals, and identification of amyloid plaques in retinas from human Alzheimer's disease patients, even before clinical symptoms allow
Conclusions
This study provides the first demonstration of Aβ plaques in postmortem retinas from suspected and definite AD patients that reflected AD brain pathology. In addition, systemic administration of curcumin to AD mice resulted in specific in vivo labeling of retinal Aβ plaques. This novel approach enabled noninvasive and high-resolution monitoring of individual retinal Aβ plaques in live AD mice. Finally, curcumin-visualized retinal plaques were shown to decrease in number and size following
Competing interests statement
The authors declare that no conflict of interest exists.
Acknowledgments
We thank Drs. J.Y. Hwang, Y. Kohanzadeh, A.G. Nowatzyk, K.V. Ramanujan and K. Wawrowsky for useful discussions and imaging collaboration. M.S. holds the Maurice and Ilse Katz Professorial Chair in Neuroimmunology at The Weizmann Institute of Science, Israel. This work was supported in part by the Marciano Family Foundation, the U.S. Navy Bureau of Medicine and Surgery, R01 EY13431 and M01 RR00425, the Winnick Family Foundation, and the University of Southern California Alzheimer's Disease
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