Conclusive demonstration of iatrogenic Alzheimer's disease transmission in a model of stem cell transplantation

Abstract

The risk of iatrogenic disease is often underestimated as a concern in contemporary medical procedures, encompassing tissue and organ transplantation, stem cell therapies, blood transfusions, and the administration of blood-derived products. In this context, despite the prevailing belief that Alzheimer's disease (AD) manifests primarily in familial and sporadic forms, our investigation reveals an unexpected transplantable variant of AD in a preclinical context, potentially indicating iatrogenic transmission in AD patients. Through adoptive transplantation of donor bone marrow stem cells carrying a mutant human amyloid precursor protein (APP) transgene into either APP-deficient knockout or normal recipient animals, we observed rapid development of AD pathological hallmarks. These pathological features were significantly accelerated and emerged within 6-9 months post transplantation and included compromised blood-brain barrier integrity, heightened cerebral vascular neoangiogenesis, elevated brain-associated β-amyloid levels, and cognitive impairment. Furthermore, our findings underscore the contribution of β-amyloid burden originating outside of the central nervous system to AD pathogenesis within the brain. We conclude that stem cell transplantation from donors harboring a pathogenic mutant allele can effectively transfer central nervous system diseases to healthy recipients, mirroring the pathogenesis observed in the donor. Consequently, our observations advocate for genomic sequencing of donor specimens prior to tissue, organ, or stem cell transplantation therapies, as well as blood transfusions and blood-derived product administration, to mitigate the risk of iatrogenic diseases.

Keywords: Alzheimer’s disease; CNS disorders; blood transfusion; iatrogenic diseases; megakaryocytes derived amyloid beta; mouse behaviour analysis; organ transplant; prion diseases; protein misfolding; stem-cell transplant.

Graphical abstract

Figure 1

Successful reconstitution of the donor BMC population in the recipient (A) Representative overlaid scatterplot of splenocytes from a CD45.1+ donor (blue, AD or WT) and a CD45.2+ recipient mouse (red, APP KO) before bone marrow reconstitution. (B) Representative scatterplot of PBMCs from a CD45.2+ APP KO mouse, reconstituted with CD45.1+ bone marrow from a donor mouse (AD or WT), 2 months post transplant. The recipient APP KO mouse is 91.5% CD45.1 positive, indicating successful reconstitution.

Figure 2

Bone marrow transfer from AD mice causes cognitive deficits in APP KO recipient mice APP KO recipient mice with a successful reconstitution of donor BMCs were assessed for their cognitive status, using tests designed for the analysis of different aspects of memory. The data were pooled from two different trials unless stated otherwise and are represented as the mean ± standard deviation. (A–D) Open-field test (OFT). (A) APP KO mice that received BMCs from WT mice (WT→APP KO) spent significantly less time in the center of the field compared to APP KO mice that received BMCs from AD mice (AD→APP KO). (B) There was no significant difference in the distance traveled in the open field between WT→APP KO mice and AD→APP KO mice. (C) AD→APP KO mice have a significantly higher number of entries in the central squares of the field than WT→APP KO mice. (D) Representative track plots from the OFT. WT→APP KO mice spend less time exploring the open center of the test arena while AD→APP KO mice explore the entire field indiscriminately. Each plot in the figure represents the track from a different mouse. (A–C) An unpaired t test was used to calculate statistical significance (p ≤ 0.05). Data from two studies were pooled; AD→APP KO, n = 8 males, n = 10 females; and WT→APP KO, n = 7 males, n = 7 females. (E) Spontaneous alternation (Y maze) test. A significantly high POA was observed in the WT→APP KO mice versus the AD→APP KO mice. An unpaired t test was used to calculate statistical significance (p ≤ 0.05). AD→APP KO, n = 8 males, n = 10 females; and WT→APP KO, n = 7 males, n = 7 females. (F) Contextual fear conditioning test (FC). WT→APP KO mice exhibited high freezing percentages compared to AD→APP KO mice. Due to technical issues, data for this test were collected only from one trial. An unpaired t test was used to calculate statistical significance (p ≤ 0.05). Data for this test are only from the first trial due to issues with equipment at the time of the experiment. AD→APP KO, n = 4 males, n = 4 females; and WT→APP KO, n = 3 males, n = 3 females. (G and H) RAWM. Both (G) latency time and (H) the total number of errors were measured. WT→APP KO mice showed a significant decrease in the latency time and decreased number of errors when comparing results from test day 1 and test day 5. No significant differences between test day 1 and test day 5 were seen in the AD→APP KO. A paired t test was used to calculate significance (p ≤ 0.05). AD→APP KO, n = 8 males, n = 10 females; and B6/SJL.BM→APP KO, n = 7 males, n = 7 females.

Figure 3

Bone marrow transfer from AD mice causes cognitive deficits in certain aspects of memory in WT recipient mice (A) Y maze. A significantly high POA was observed in WT mice as compared to AD animals. AD→WT mice were tested and shown to have no significant difference in percentage alternation as compared to AD mice; however, they showed a significantly lower POA as compared to the WT mice. Data is shown as the mean ± SD and is representative of AD→WT, n = 8 (five males, three females) and WT; n = 11 (six males, five females); and AD, n = 13 (6 males, 7 females). An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to calculate statistical significance (p ≤ 0.05). (B and C) RAWM. Both: (B) latency time and (C) the total number of errors were measured. B6/SJL control mice showed a significantly lower latency time and number of errors on day 5 compared to day 1. Although AD→WT animals showed a significantly lower latency time, like the AD mice, they showed no significant difference in the number of errors between test day 1 and test day 5. AD mice showed no difference in the latency time between day 1 and day 5. The data in (B) and (C) are expressed as the mean of the latency time or the total number of errors in a single trial per day by individual animals in each group. Since the difference was being noted on day 1 versus day 5 within each group, a paired t test was used to calculate significance (p ≤ 0.05).

Figure 4

AD pathology is seen in the brains of APP KO mice reconstituted with AD bone marrow (A) Micrographs show the immunostaining and confocal imaging of the cortical region of WT→APP KO and AD→APP KO mouse brains. The expression levels of CD105 (green), DAPI (cyan), Aβ (red), and CD31 (blue) are shown where Aβ and CD105 are significantly higher in AD→APP KO mice compared to WT→APP KO mice. Data are shown as mean ± SD of percentage area of expression. Unpaired Student’s t test was used to calculate significance (p ≤ 0.05). (B) Qualitative IHC shows the expression of CD105 (green), Aβ (red), and occludin (blue). The occludin expression is inverse to that of CD105 and Aβ, and Aβ is seen co-localizing with CD105. (C) Qualitative western blotting analysis indicates the presence of mutant human APP protein in AD→APP KO animals and an absence of it in the WT→APP KO. VEGFa is expressed higher in AD→APP KO (n = 5) compared to WT→APP KO (n = 4). Expression levels of TJP, ZO1, are higher in WT→APP KO (n = 7) compared to AD→APP KO (n = 8). Blots shown here are from pooled samples from the animals in the groups to be representative of the group. Histograms show expression levels of proteins in individual animals. Unpaired Student’s t test was used to calculate significance (p ≤ 0.05).

Figure 5

Presence of amyloid plaques in AD→WT mice suggests establishment of AD pathology in WT mice through a bone marrow transplant (A) Micrographs are representative of the cortical region of mouse brains. (B) Congo red staining indicates the establishment of amyloid plaques in AD→WT mice, which is seen to be significantly higher as compared to WT and similar to the levels seen in AD animals. Each data point in the histograms corresponds to data from individual mice in each group, where n = 6 for WT, n = 8 for AD, and n = 8 for AD→WT. Data is represented as mean ± SD. Ordinary one-way ANOVA was used to calculate significance (p ≤ 0.05).