Jörg Hanrieder (Presenter)
University of Gothenburg
Bio: Jörg Hanrieder received training in organic and analytical chemistry at Leipzig University, Germany. In 2006, he moved on to a PhD in Analytical Neurochemistry at Uppsala University, Sweden. After his PhD in 2010 he obtained a prestigious PostDoc grant from the Swedish government to work with Prof Andrew Ewing at Chalmers in Gothenburg, Sweden. In 2014 he received a Marie Curie young investigator grant and is currently junior faculty at the Dept of Neuroscience and Physiology at the University of Gothenburg. In addition, he holds an honorary senior researcher position at the Institute of Neurology at UCL in London.
Authorship: Jörg Hanrieder (1,2,3), Wojciech Michno (1), and Stina Syvänen (4)
(1) University of Gothenburg, Mölndal, Sweden; (2) University College London, UK; (3) Chalmers University of Technology, Gothenburg, Sweden; (4) Uppsala University, Uppsala, Sweden
| Short Abstract The pathological mechanisms underlying Alzheimer’s disease are still not understood. The disease is characterized by accumulation and aggregation of amyloid peptides into extracellular plaques. The factors that promote neurotoxic amyloid peptide aggregation remain elusive. In the present study, multimodal (SIMS and) MALDI imaging was used to study individual amyloid plaques in brain tissue from in brain sections of transgenic AD mice (tgARCSWE) in oder to elucidate the plaque associated chemical microenvironment. PCA image analysis was used to interrogate the IMS data set for identifying anatomical features based on their chemical identity. Statistics on spectral data of regions of interest reveal brain region specific changes in amyloid peptide pathology and lipid content. This was further verified using immunohistochemistry and laser micro dissection and MALDI MS of plaque extracts. |
Long Abstract
Alzheimer’s disease is the most common neurodegenerative disorder affecting 12% over 65 (1). The exact mechanisms underlying AD pathogenesis are still not fully understood, significantly hampering the development of therapeutic treatment strategies. In AD, cognitive decline has been linked to formation of β-amyloid (Aβ) deposits as senile plaques as well as intracellular neurofibrillary tangles comprised of hyper-phosphorylated tau protein (2). Aβ aggregation has been suggested as a possibly critical, early inducer driving the disease progression. Over the years several transgenic mouse models have been developed, since the neuropathology in genetic- and sporadic AD is similar with respect to protein accumulation (1). The arctic and swedish mutation (ARCSWE) of amyloid precursor protein (APP) results in significant increase of neurotoxic Aβ peptides and fibrils (3).
Changes in amyloid peptide truncation and plaque associated neuronal lipid species have been implicated with proteopathic mechanisms in AD (1,4).Current biochemical methods however lack the neccessary molecular specificity to study plaque chemistry, highlighting the need to develop and employ new bioanalytical techniques such as imaging mass spectrometry (IMS) (5).
The aim of this study was therefore to employ SIMS and MALDI based multimodal imaging mass spectrometry to probe Aβ plaque pathology in tgARCSWE mice with particular focus on associated neuronal lipid species and Aβ peptide truncation.
Transgenic C57BL/6-CBA-F1 male mice carrying the swedish double mutation alone (K670M, N671L) and the Arctic mutation (E693G) of human APP were studied. Lipid changes were examined in adult mouse brain (18 month) using ToF-SIMS and MALDI imaging on fresh frozen coronal cryosections at two different bregma (CPu and Hippocampus).
Here, tissue cryosections from 18 month old tgARCSWE mice that displayed extensive plaque pathology were analyzed using MALDI and SIMS imaging of individual Aβ plaques. MALDI imaging analysis revealed the Aβ peptide chemistry in these plaques. Here, different truncations (Aβ3pE-40 and Aβ1-40) were observed, including the pyroglutamate truncation, which is considered very neurotoxic and promotes extensive Aβ oligomerization (6). High resolution (300nm) SIMS analysis of the same tissue section revealed localization of phosphocholine (PC) lipids to individual plaques as indicated by the PC-headgroup (m/z 184.09). In addition, characteristic localization of cholesterol to the center of the plaque was observed. This is of particular interest, since cholesterol is transported by APOE, and the APOE4 allele is a major genetic risk factor for AD (4). Moreover, cholesterol has previously been identified as amyloid- membrane anchor, promoting Aβ aggregation (7).
The IMS staining experiments were complemented with immunohistochemistry (IHC) towards Aβ on the same section to verify the Aβ identity of these plaques in general. In order to determine the fibrillization state of these plaques, a hyper-spectral imaging paradigm was developed that allowed multiplexed staining with luminescent conjugated oligothiophene (LCO) amyloid staining probes and fluorescent-labeled anti-amyloid antibodies. LCO probes are structurally based on thioflavin, which is commonly used for general amyloid plaque staining (8). These LCO’s exhibit specificity to different amyloid aggregation states. Structural- and immuno-staining of Aβ fibrils was performed on the same tissue section, following MALDI and SIMS imaging. Here two LCO’s probes were used that are stimulated at different wavelengths including h-FTAA and q-FTAA. Q-FTAA responds only to mature Aβ amyloid fibrils, whereas h-FTAA binds to mature amyloid fibrils and responds in addition to early pre-fibrillar states (diffuse plaques).
This LCO double staining strategy facilitated to identify diffuse and more mature plaques based on their degree of fibrillization. This can in turn be correlated with chemical properties determined by imaging MS.
Finally, double positive plaques were excised using laser microdissection (LMPC). The collected plaques were subjected to formic acid extraction in order to retrieve the individual polypeptide species. The plaque extracts were then analyzed using mass spectrometry. Here, the various C-terminal Aβ species were characterised, validating the imaging MS and fluorescence staining experiments. A manuscript with these results is currently in preparation.
In conclusion, IMS and LCO based multimodal imaging is therefore a promising approach to interrogate chemical plaque pathology in Alzheimer’s disease.
References & Acknowledgements:
References:
(1) Selkoe, D.J. and Schenk D., Annu Rev Pharmacol Toxicol, 2003.43: p. 545-84
(2) Thal D.R. et al., Neurology, 2002. 58(12): p. 1791-800
(3) Lord, A., et al., Neurobiol Aging, 2006. 27(1): p. 67-77
(4) Di Paolo G. and Kim TW, Nat Rev Neurosci, 2011, 12(5): p.284-296
(5) Hanrieder J. et al. ACS Chem Neurosci, 2014, 5(7): p.568-575
(6) Hardy, J. and Selkoe, D.J. Science 2002, 297. 353-56
(7) Barrett, P.J., Song, Y., Van Horn, W.D., et al., Science 2012, 336. 1168-71
Acknowledgements: The Swedish Research Council is acknowledged for financial support (Young Investigator Grant (JH))
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