Mapping the Spatial Distribution of Fibrillar Polymorphs in Human Brain Tissue

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder defined by the progressive formation and spread of fibrillar aggregates of Aβ peptide and tau protein. Polymorphic forms of these aggregates may contribute to disease in varying ways since different neuropathologies appear to be associated with different sets of fibrillar structures and follow distinct pathological trajectories that elicit characteristic clinical phenotypes. The molecular mechanisms underlying the spread of these aggregates in disease may include nucleation, replication, and migration all of which could vary with polymorphic form, stage of disease, and region of brain. Given the linkage between mechanisms of progression and distribution of polymorphs, mapping the distribution of fibrillar structures in situ has the potential to discriminate between mechanisms of progression. However, the means of carrying out this mapping are limited. Optical microscopy lacks the resolution to discriminate between polymorphs in situ, and higher resolution tools such as ssNMR and cryoEM require the isolation of fibrils from tissue, destroying relevant spatial information. Here, we demonstrate the use of scanning x-ray microdiffraction (XMD) to map the locations of fibrillar polymorphs of Aβ peptides and tau protein in histological thin sections of human brain tissue. Coordinated examination of serial sections by immunohistochemistry was used to aid in the interpretation of scattering patterns and to put the observations in a broader anatomical context. Scattering from lesions in tissue shown to be rich in Aβ fibrils by immunohistochemistry exhibited scattering patterns with a prototypical 4.7 Å cross-β peak, and overall intensity distribution that compared well with that predicted from high resolution structures. Scattering from lesions in tissue with extensive tau pathology also exhibited a 4.7 Å cross-β peak but with intensity distributions that were distinct from those seen in Aβ-rich regions. In summary, these observations demonstrate that XMD is a rich source of information on the distribution of fibrillar polymorphs in diseased human brain tissue. When used in coordination with neuropathological examination it has the potential to provide novel insights into the molecular mechanisms underlying disease.

Small-angle x-ray microdiffraction from fibrils embedded in tissue thin sections

“…Scattering from the non-fibrillar tissue constituents is removed from fibrillar scattering within the plaque by assuming that the scattering of the tissue constituents within the lesion is the same as in a neighboring region devoid of fibrils. Since scattering from the underlying mica substrate does not require scaling, we carried out subtraction according to I = (I l - I b ) - a (I t - I b ) where I l is the observed scattering from the lesion; I t that observed from proximal tissue; I b background observed from scattering from mica devoid of tissue (Bashit et al, 2022). The scale factor, a, is chosen to minimize the difference between plaque and background intensities in the range 1.6 < q < 2.0 Å -1 , where all evidence has indicated the fibrillar scattering is weak.…”

“…Analysis of the equatorial scattering can provide information about the diameter and shape of the fibrils and the arrangement of material around the fibril within the tissue (Roig Solvas and Makowski, 2017). Comparison of the shape of the 4.7 Å reflection with that predicted from high resolution structures obtained by cryoEM studies of isolated or in vitro -assembled material may provide identification of the precise structure within the scattering volume (Bashit et al, 2022). In this report, we limit our analysis to the small-angle regime.…”

Small-angle X-ray microdiffraction from fibrils embedded in tissue thin sections

“…Five background patterns are selected from regions adjacent to potential lesions and averaged. Since scattering from the underlying mica substrate does not require scaling, we carried out subtraction according to I = (I l À I b ) À a(I t À I b ), where I l is the observed scattering from the lesion, I t is that observed from proximal tissue and I b is the background observed from scattering of mica devoid of tissue (Bashit et al, 2022). In our experience I b is relatively invariant over the millimetre length scales of individual mica films, but may vary over centimetre length scales.…”

“…Analysis of the equatorial scattering can provide information about the diameter and shape of the fibrils and the arrangement of material around the fibril within the tissue (Roig-Solvas & Makowski, 2017). Comparison of the shape of the 4.7 A ˚reflection with that predicted from high-resolution structures obtained by cryoEM studies of isolated or in vitroassembled material may provide identification of the precise structure within the scattering volume (Bashit et al, 2022). In this report, we limit our analysis to the small-angle regime.…”

Label-free and reference region-free X-ray cross-β index for quantifying protein aggregates of neurodegenerative diseases

“…The cross-β structure composed of arrays of β-sheets running parallel to the long axis of the biomarker aggregates and the structure is absent for functional protein monomers [28]. The presence of cross-β rich aggregates were reported previously by X-ray microdiffraction studies for ex-vivo human brain tissues of patients with Lewy bodies of PD [29], amyloid plaques of AD [28], tau deposits of Down syndrome and frontotemporal lobar degeneration (FTD) [30] and glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) [29] (Table S1 in supporting information). The prior studies were limited to the qualitative distinction of healthy and diseased postmortem tissues based on the absence or presence of X-ray cross-β signals of matured biomarker deposits.…”