GM-CSF upregulated in rheumatoid arthritis reverses cognitive impairment and amyloidosis in Alzheimer mice

Abstract

Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). While it has been commonly assumed that RA patients' usage of non-steroidal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of AD, NSAID clinical trials have proven unsuccessful in AD patients. To determine whether intrinsic factors within RA pathogenesis itself may underlie RA's protective effect, we investigated the activity of colony-stimulating factors, upregulated in RA, on the pathology and behavior of transgenic AD mice. 5 microg bolus injections of macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factors (M-CSF, G-CSF, or GM-CSF) were administered unilaterally into the hippocampus of aged cognitively-impaired AD mice and the resulting amyloid load reductions determined one week later, using the artificial cerebrospinal fluid-injected contralateral sides as controls. G-CSF and more significantly, GM-CSF reduced amyloidosis throughout the treated brain hemisphere one week following bolus administration to AD mice. 20 daily subcutaneous injections of 5 microg of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) were administered to balanced cohorts of AD mice after assessment in a battery of cognitive tests. Reductions in amyloid load and improvements in cognitive function were assessed. Subcutaneous GM-CSF administration significantly reduced brain amyloidosis and completely reversed the cognitive impairment, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, recombinant human GMCSF, in elderly leukopenic patients, suggest that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

Fig. 1

Intrahippocampal injection of GM-CSF (left) and artificial cerebrospinal fluid (aCSF) (right). a) Representative coronal tissue cryo-sectioned at 14 µ m and stained with MabTech α-Aβ/Alexa 546. Image is a montage of about 145 pictures taken at 10×. White spots indicate amyloid plaque immunolabeling (see Supplementary Fig. 4 online for representative montaged sections of all 4 mice). b) Significant overall plaque reductions seen in all 4 plaque parameters measured from 5 quantified sections per mouse (n = 4 mice). Error bars are ± Standard Error of the Mean. (Area: p < 1.11E-07; Perimeter: p < 1.41E-06; Feret Diameter: p < 2.36E-09; Integrated Density: p < 1.11E-07).

Fig. 2

Behavioral analysis following daily subcutaneous GM-CSF injections. a) Standard RAWM errors prior to treatment. The final block and overall performance of the Tg and NT mice during 8 days of consecutive daily pretreatment testing in the RAWM maze. The data was analyzed in four 2-day blocks and overall (Blocks 1–4). (*p < 0.02 or higher significance). b) Standard RAWM errors after treatment. Tg control mice (n = 6) show substantial impairment on working memory trials T4 and T5 compared to NT control mice (n = 8) in individual blocks of testing (upper), and over all 4 days of testing (lower). GM-CSF-treated Tg mice (n = 5) performed as well as or better than NT control mice on working memory trials T4 and T5 during individual blocks and over all. GM-CSF-treated NT mice (n = 9) performed similarly to or slightly better than NT controls (Note significantly better performance of NT+GM-CSF group versus NT group for T4 of Block 1), although this effect was not significant overall. (**p < 0.02 or higher significance versus all other groups; ^†p < 0.02 or higher significance versus Tg+GM-CSF and NT+GM-CSF). c) Cognitive Interference Task. Overall (4 Days) Tg control mice are impaired compared to NT mice on all four cognitive measures assessed. GM-CSF-treated Tg mice exhibited significantly better 3-trial recall (A1–A3) and delayed recall (A5) compared to Tg controls and performed similarly to NT mice in all four cognitive measures. GM-CSF treatment of NT mice did not result in significantly better performance compared to NT controls, although trends for a beneficial GM-CSF effect in NT mice were evident overall. (*Tg significantly different from NT+GM-CSF, **Tg significantly different from all other groups). d) Cognitive Interference Task. Proactive Interference testing (First 2 days). GM-CSF-treated Tg mice performed significantly better than Tg controls and equally to NT and GM-CSF-treated NT mice.

Fig. 3

Amyloid deposition in subcutaneous GM-CSF-injected mice. a–d) Photomicrographs of coronal 5 µm paraffin-embedded sections immunolabelled with anti-Aβ antibody (clone 4G8) in entorhinal cortex (EC) and hippocampus (H). Pictures are representative of amyloid load closest to the mean of the GM-CSF- or saline-treated Tg groups. Scale bar = 50 µm. e) Percent of amyloid burden from the average of five 5 µm sections (150 µm apart) through both anatomic regions of interest (hippocampus and entorhinal cortex) per mouse of GM-CSF-treated (n = 5) versus saline-treated (n = 6). Entorhinal cortex (*p < 0.026), and hippocampus (p = 0.12).

Fig. 4

Microglial immunostaining in subcutaneous GM-CSF-injected mice. a–d) Photomicrographs of coronal 5 µm paraffin-embedded sections immunolabeled with Iba-1 antibody in entorhinal cortex (EC) and hippocampus (H). Pictures are representative of Iba-1 immunolabeling closest to the mean of the GM-CSF- or saline control-treated groups. Scale bar = 50 µm. e) Percent of Iba1 burden from the average of five 5 µm sections (150 µm apart) through both anatomic regions of interest per mouse of GM-CSF-treated (n = 5) versus saline-treated (n = 6). Hippocampus (p < 0.02), entorhinal cortex (p < 0.05).

Fig. 5

Synaptophysin immunostaining in subcutaneous GM-CSF-injected mice. a–d) Photomicrographs of coronal 5 µm paraffin-embedded sections immunolabeled with antisynaptophysin antibody. Pictures are representative of synaptophysin immunolabeling closest to the mean of the GM-CSF- or saline control-treated groups. Scale bar = 50 µm. e) Percent of synaptophysin immunoreactivity from the average of 5 sections per mouse of GM-CSF-treated (n = 5) versus saline control-treated (n = 6). CA1 (p < 0.0013), CA3 (p < 0.0023).