Suppressed retinal degeneration in aged wild type and APPswe/PS1ΔE9 mice by bone marrow transplantation.

Alzheimer's disease (AD) is an age-related condition characterized by accumulation of neurotoxic amyloid β peptides (Aβ) in brain and retina. Because bone marrow transplantation (BMT) results in decreased cerebral Aβ in experimental AD, we hypothesized that BMT would mitigate retinal neurotoxicity through decreased retinal Aβ. To test this, we performed BMT in APPswe/PS1ΔE9 double transgenic mice using green fluorescent protein expressing wild type (wt) mice as marrow donors. We first examined retinas from control, non-transplanted, aged AD mice and found a two-fold increase in microglia compared with wt mice, prominent inner retinal Aβ and paired helical filament-tau, and decreased retinal ganglion cell layer neurons. BMT resulted in near complete replacement of host retinal microglia with BMT-derived cells and normalized total AD retinal microglia to non-transplanted wt levels. Aβ and paired helical filament-tau were reduced (61.0% and 44.1% respectively) in BMT-recipient AD mice, which had 20.8% more retinal ganglion cell layer neurons than non-transplanted AD controls. Interestingly, aged wt BMT recipients also had significantly more neurons (25.4%) compared with non-transplanted aged wt controls. Quantitation of retinal ganglion cell layer neurons in young mice confirmed age-related retinal degeneration was mitigated by BMT. We found increased MHC class II expression in BMT-derived microglia and decreased oxidative damage in retinal ganglion cell layer neurons. Thus, BMT is neuroprotective in age-related as well as AD-related retinal degeneration, and may be a result of alterations in innate immune function and oxidative stress in BMT recipient mice

Aβ and PHF-tau are reduced in retina of APP<i>swe</i>-PS1ΔE9 BMT recipient mice.

<p><b>A</b>: Representative photomicrographs of Aβ deposition in non-transplanted, age-matched APP<i>swe</i>-PS1ΔE9 control retina (top, AD No Tx) or APP<i>swe</i>-PS1ΔE9 that received BMT (bottom, AD BMT) stained with anti-Aβ antibody and visualized with Cy3-conjugated secondary antibody (red). Region of inset is indicated by arrows. Scale bar  = 50 µm. <b>B</b>: Quantitative analysis of Aβ immunofluorescence using a standardized digital thresholding protocol demonstrated significant reduction in retinal Aβ in BMT APP<i>swe</i>-PS1ΔE9 mice compared with non-transplanted APP<i>swe</i>-PS1ΔE9 control mice (***<i>P</i><0.001, n = 6, student's <i>t</i> test). <b>C</b>: Representative photomicrographs of PHF-tau immunofluorescence in retinal ganglion cell layer (RGCL) (arrows) of non-transplanted, age-matched control APP<i>swe</i>-PS1ΔE9 mice (top, AD No Tx) compared with APP<i>swe</i>-PS1ΔE9 BMT recipients (bottom, AD BMT). Nuclei were counterstained with DAPI (blue). Scale bar  = 30 µm. <b>D</b>: Quantitative analysis of PHF-tau immunofluorescence using a standardized digital thresholding protocol demonstrated significant reduction of PHF-tau in APP<i>swe</i>-PS1ΔE9 BMT-recipients compared with non-transplanted controls (*<i>P</i><0.05, n = 6, one-way ANOVA analysis with Bonferroni <i>post</i> test).</p

Age-related RGCL neurodegeneration is mitigated by BMT in wt and APP<i>swe</i>-PS1ΔE9 mice.

<p>NeuN⁺ RGCL neuron density was compared between 13-month-old BMT recipient mice and non-transplanted young (5 mo) and age-matched (13 mo) wt and APP<i>swe</i>-PS1ΔE9 mice and presented as percent of 5-month-old wt non-transplanted controls. There was a significant age-dependent reduction in neuron density that was partially rescued in wt and APP<i>swe</i>-PS1ΔE9 BMT recipient mice. ***<i>P</i><0.001, ^#<i>P</i><0.05, ^##<i>P</i><0.01, n = 6–10, two-way ANOVA followed by Bonferroni <i>post</i> test.</p

MHC class II is up-regulated in BMT-derived retinal microglia.

<p><b>A</b>: Representative photomicrographs of microglia in the retina of a 13-month-old non-transplanted wt mouse stained with anti-Iba-1 antibody and visualized with Cy3-conjugated secondary antibody (red). The endogenous Iba-1⁺ microglial cells do not express detectable MHC class II by immunofluorescence stains (blue, overlay with Cy3 fluorescence) in untreated (No Tx) wt retina. <b>B</b>: Representative photomicrographs of retinal sections from a 13-month-old wt mouse transplanted with GFP⁺ BM cells. Immunofluorescence staining demonstrates that the Iba-1⁺ microglia (red) are almost completely derived from the BMT (green GFP⁺ cells). GFP⁺ cells were strongly immunoreactive for MHC class II (blue). The overlay of the confocal microscope images indicates co-expression of GFP (green), Iba-1 (red) and MHC class II (blue) in retina. Scale bar  = 30 μm. <b>C</b>: Quantification of MHC class II immunofluorescence in microglia shows significantly increased expression in BM-derived cells compared with the endogenous microglia. ***<i>P</i><0.001, n = 6, student's <i>t</i> test. <b>D</b>: Confocal analysis of sections demonstrates association of BM-derived microglia (GFP⁺, green, arrow) and Aβ deposits (red). High magnification of inset is shown on the lower panels. Scale bar: 20 µm. <b>E</b>: 3D-Confocal image analysis of intracellular Aβ in BM-derived microglia. The overlay of confocal images reveals Aβ deposition (red) within GFP⁺ BM-derived microglia (green, arrow). High magnification of inset is shown on the right panels. Scale bar: 20 µm.</p

Increased microglia density in experimental AD is mitigated by BMT.

<p><b>A</b>: Representative retinal cryosections from wt (left) and APP<i>swe</i>-PS1ΔE9 mice (right) were stained with anti-Iba-1 antibody and visualized with Cy3-conjugated secondary antibody. Ramified microglia were primarily identified in ganglion cell layer (gcl), inner plexiform layer (ipl) and outer plexiform layer (opl) in wt and APP<i>swe</i>-PS1ΔE9 retinas. <b>B</b>: Unbiased stereologic analysis revealed increased microglia density in control (untreated) APP<i>swe</i>-PS1ΔE9 retina compared with wt control mice (*<i>P</i><0.05, n = 7–9, student's <i>t</i> test). <b>C</b>: Average Iba-1⁺ microglia density in the different retinal layers. Significantly higher numbers of Iba-1⁺ microglia were noted in gcl, ipl and opl in APP<i>swe</i>-PS1ΔE9 mice (*<i>P</i><0.05, ***<i>P</i><0.001, n = 7–9, two-way ANOVA followed by Bonferroni <i>post</i> test.) Confocal images from wt (<b>D</b>) and APP<i>swe</i>-PS1ΔE9 (<b>E</b>) retinas immunostained with Iba-1 (red) demonstrate that BM-derived GFP⁺ cells (green) exhibited ramified microglia morphology and had near-uniform expression of Iba-1. In both wt and APP<i>swe</i>-PS1ΔE9 BMT recipient mice, host microglia were almost completely replaced with BM-derived cells. <b>F</b>: Unbiased stereologic quantitation of retinal microglia engraftment revealed 91.2±4.0% of wt and 73.2±16.0% of APP<i>swe</i>-PS1ΔE9 Iba-1⁺ microglia were BM derived (GFP⁺). <b>G</b>: Microglia density in retina of wt BMT recipients was not different from non-transplanted wt mice, but BMT in APP<i>swe</i>-PS1ΔE9 mice normalized microglia density to wt levels. Data are percent of wt Iba-1⁺ cells/mm² in non-transplanted APP<i>swe</i>-PS1ΔE9 untreated mice (AD no Tx) or APP<i>swe</i>-PS1ΔE9 and wt BMT recipients (*<i>P</i><0.05, **<i>P</i><0.01, n = 6–10, one-way ANOVA followed by Bonferroni <i>post</i> test). Scale bars  = 30 μm.</p

Experimental design.

<p>Control mice included wt and APP<i>swe</i>-PS1ΔE9 that received no irradiation or BMT and were euthanized at 5 and 13 months of age for analysis of retinal pathology. Additional control wt and APP<i>swe</i>-PS1ΔE9 mice received head only (HO) irradiation (XRT) at 5 months of age and were euthanized at 13 months of age for analysis of retinal pathology. Experimental groups included 5-month-old wt and APP<i>swe</i>-PS1ΔE9 mice that received lethal (10.5 Gy) whole body (WB) irradiation followed 24 hours later by retroorbital venous plexus injection of whole bone marrow from GFP-expressing wt mice and were then euthanized at 13 months of age for analysis of retinal pathology. Arrows mark the time points of generation and analysis of the control or treated mice.</p

BMT mediates neuroprotection of RGCL neurons in APP<i>swe</i>-PS1ΔE9 and wt mice.

<p><b>A–C</b>: RGCL neurons were identified using anti-NeuN antibody and visualized with Cy3-conjugated secondary antibody in 13-month-old APP<i>swe</i>-PS1ΔE9 and wt mice. Representative photomicrographs of NeuN⁺ RGCL neurons in control (A, top) and BMT-recipient (A, bottom) APP<i>swe</i>-PS1ΔE9 mice demonstrate neuroprotective effects of BMT through preservation of RGCL neurons (B) and inner retinal (NFL+RGCL+IPL) thickness (C). <b>D–E</b>: Representative photomicrographs of NeuN⁺ RGCL neurons in 13-month-old control (D, top) and BMT-recipient (D, bottom) wt mice also demonstrate neuroprotective effects of BMT through preservation of RGCL neurons (E). <b>F</b>: No effects on retinal thickness in wt recipients compared with the wt controls. *<i>P</i><0.05, n = 6–10, student's <i>t</i> test. Scale bar  = 50 μm.</p

RGCL neuroprotection is not due to effects of high dose cranial irradiation alone.

<p><b>A</b>: Representative photographs of control mice (no Tx) and mice that received head only irradiation (HO-XRT) demonstrate effects of irradiation on coat color and confirm radiation exposure in HO-XRT mice (top). Representative photomicrographs of NeuN⁺ neurons (red) in RGCL stained with NeuN antibody and visualized with Cy3-conjugated secondary antibody show a mild reduction in neuron density 8 months after HO-XRT (bottom). Scale bar  = 50 μm. <b>B</b>: Quantification of neuron density depicted as a percent of age-matched, non-irradiated wt controls demonstrates there is a mild reduction in RGCL neurons as a result of high dose irradiation without BMT in wt and APP<i>swe</i>-PS1ΔE9 mice, thus eliminating the possibility that high dose cranial irradiation underlies the neuroprotective effects of BMT.</p

BMT results in reduced RGCL oxidative stress in aged wt and APP<i>swe</i>-PS1ΔE9 mice.

<p><b>A:</b> Immunofluorescence stains for 8-OHdG (red), an indicator of oxidative stress, are shown in representative retinal cross-sections from age-matched wt (left column) or APP<i>swe</i>-PS1ΔE9 (right column) mice that received no BMT transplant (top row) or BMT (bottom row). 8-OHdG immunofluorescence is primarily detected in RGCL neurons in non-transplanted wt and APP<i>swe</i>-PS1ΔE9 mice in a diffuse, perikaryal pattern. However, only focal, punctate immunostaining was observed in retinas from wt and APP<i>swe</i>-PS1ΔE9 mice that received BMT. Arrows indicate regions highlighted in insets. Scale bar  = 20 μm. <b>B:</b> Quantification of 8-OHdG immunofluorescence relative intensity in RGCL neurons confirmed a significant reduction in 8-OHdG immunostaining in wt and APP<i>swe</i>-PS1ΔE9 mice that received BMT compared with non-transplanted controls, respectively. ***<i>P</i><0.001, n = 6–10, two-way ANOVA followed by Bonferroni <i>post</i> test.</p

Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

A complex therapeutic challenge for Alzheimer’s disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid β (Aβ) peptides. One potential target is selective suppression of microglial prostaglandin E2 receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2−/−) into lethally irradiated 5-month-old wild-type or APPswe-PS1ΔE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2−/− microglia was more efficient in APPswe-PS1ΔE9 than in wild-type mice, and APPswe-PS1ΔE9 mice that received EP2−/− BMT had increased cortical microglia compared with APPswe-PS1ΔE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Aβ plaques and potentially more neurotoxic soluble Aβ species. An additional 25% reduction in cerebral cortical Aβ burden was achieved in mice that received EP2−/− BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Aβ peptide and plaque accumulation in the cerebrum of patients with AD