Unraveling pathogenic proteins and pathways in Alzheimer disease : a focus on proteomics

Alzheimer disease (AD) is a multifactorial and complex neurodegenerative disorder. To date, different mechanisms, such as impairment of synaptic, mitochondrial and autophagic function, neuroinflammation and many more, are found to disrupt cellular homeostasis in AD brains. Despite the increased knowledge, it is still difficult to pinpoint which of these mechanisms is the main culprit driving the pathologic cascade, especially in the form of late- onset, sporadic AD, accounting for more than 95% of all patients. In this thesis, we used human-based or translational approaches to investigate which pathological alterations indeed occur in AD brains.

In Paper I, we investigated an amyloid precursor protein (APP)-derived band with a molecular weight of 20 kDa, most likely corresponding to the APP C-terminal fragment (CTF) called CTF-η, and showed that it is expressed at low levels in the human brain. However, we also noted that several antibodies directed to APP or other proteins also detects a presumably non-specific band of a similar size. In Paper II, IV and V, we explored changes in the proteome of postmortem AD brains and CSF of AD patients and App knock-in mice. In Paper II, our aim was to identify proteins and pathways that could underlie synaptic dysfunction, a pathogenic event that happens early in disease progression. We thus explored the proteome of the outer molecular layer (OML) of the dentate gyrus using mass spectrometry (MS). This region is relatively cell-free and highly enriched in synaptic connections, and more importantly receives the main input of the hippocampus called the perforant path, which is highly affected in AD pathogenesis. Our comprehensive data analysis indicates that the OML indeed exhibits presynaptic changes, which is in line with previously published reports, whereas postsynaptic density proteins were not altered. To follow-up on the hypothesis of presynaptic impairment in AD OML, using immunofluorescence, we measured the staining densities of five presynaptic proteins in sub-regions of the hippocampus in Paper III. Similarly, we found decreased staining densities of complexin-1, syntaxin-1a, synaptotagmin-1 and synaptogyrin-1 in AD OML. However, the analysis of other hippocampal sub-regions showed no significant alterations in these presynaptic proteins, except syntaxin-1a, which showed increased staining densities in AD. Although other molecular layers of hippocampus also receive the perforant path input (as well as other important inputs), it was intriguing to find that presynaptic impairment was restricted to the OML. Together, Paper II and III point out to presynaptic failure in AD hippocampus.

To further compare our proteomic findings with the published ones, in which proteome of AD-affected brain regions (e.g. temporal and frontal cortices) was analyzed, and to identify commonalities and discrepancies between the studies, in Paper IV, we performed a meta-analysis of labeled (11753 proteins and 168 cases) and label-free (4292 proteins and 632 cases) data. We found approximately 500 significantly altered proteins that were associated with pathways such as synaptic signaling, neuron and axon development, neurogenesis, cellular respiration and catabolic process, some of which are previously reported to be involved in AD pathogenesis. Additionally, seven novel proteins were found to be consistently altered in AD.

In Paper V, we studied the CSF proteome of App knock-in mice and identified alterations in several blood-brain barrier and extracellular matrix proteins, for example decorin. Furthermore, in order to explore translational changes between mouse and human CSF, we compared our findings from Paper V with the CSF proteome of human patients, reflecting different stages in AD continuum (i.e normal cognition, mild cognitive impairment and AD dementia), from a recently published study. Interestingly, decorin was significantly upregulated both in the AppNL-F/NL-F mice and in the subjects with normal cognition and Aβ-positive and tau-negative CSF levels. Additionally, this study revealed alterations in proteins that were shared in all groups and extensively associated with pathways such as cell adhesion, neurogenesis, cholesterol and lipid metabolism and acute inflammatory response.

In summary, this thesis has contributed with new knowledge on potential presynaptic failure in AD hippocampus and expanded our understanding of altered pathways that could be involved in AD pathogenesis. Future studies on this work may facilitate the development of new CSF biomarkers and therapeutic strategies for AD.

List of scientific papers

I. Hazal Haytural*, Jolanta L. Lundgren*, Tansu B. Köse, Tomàs Jordà-Siquier, Marinela Kalcheva, Mohammed Seed Ahmed, Bengt Winblad, Erik Sundström, Gaël Barthet, Lars O. Tjernberg and Susanne Frykman. Non-specific Detection of a Major Western Blotting Band in Human Brain Homogenates by a Multitude of Amyloid Precursor Protein Antibodies. Frontiers in Aging Neuroscience. 2019 Oct 9;11:273. *These authors contributed equally.
https://doi.org/10.3389/fnagi.2019.00273

II. Hazal Haytural, Georgios Mermelekas, Ceren Emre, Saket Milind Nigam, Steven L. Carroll, Bengt Winblad, Nenad Bogdanovic, Gael Barthet, Ann- Charlotte Granholm, Lukas M. Orre, Lars O. Tjernberg, and Susanne Frykman. The Proteome of the Dentate Terminal Zone of the Perforant Path Indicates Presynaptic Impairment in Alzheimer Disease. Molecular & Cellular Proteomics. 2020 Jan;19(1):128-141.
https://doi.org/10.1074/mcp.RA119.001737

III. Hazal Haytural*, Tomás Jordá-Siquer*, Bengt Winblad, Christophe Mulle, Lars O. Tjernberg, Ann-Charlotte Granholm, Susanne Frykman, Gaël Barthet. Distinctive alteration of presynaptic proteins in the outer molecular layer of the dentate gyrus in Alzheimer’s disease. *These authors contributed equally. [Manuscript]

IV. Hazal Haytural, Rui Benfeitas, Sophia Schedin-Weiss, Erika Bereczki, Melinda Rezeli, Richard Unwin, Eric B. Dammer, Eric C.B. Johnson, Nicholas T. Seyfried, Bengt Winblad, Betty Timjs, Pieter J. Visser, Susanne Frykman, Lars O. Tjernberg. Insights into the changes in the proteome of Alzheimer disease elucidated by a meta-analysis. [Manuscript]

V. Richeng Jiang, Una Smailovic, Hazal Haytural, Robert Mihai Haret, Ganna Shevchenko, Betty Tijms, Johan Gobom, Henrik Zetterberg, Bengt Winblad, Susanne Frykman, Vesna Jelic, Jonas Bergquist, Pieter Jelle Visser, Per Nilsson. Autophagy-activating extracellular matrix protein decorin is increased in CSF of App knock-in mice and early stage of Alzheimer. [Manuscript]