https://doi.org/10.1016/j.expneurol.2017.10.017

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Title

Critical appraisal of pathology transmission in the α-synuclein fibril model of Lewy body disorders

Citation and DOI for published article

Nouraei, N., Mason, D. M., Miner, K. M., Carcella, M. A., Bhatia, T. N., Dumm, B. K., Soni, D., Johnson, D. A., Luk, K. C., & Leak, R. K. (2018). Critical appraisal of pathology transmission in the α-synuclein fibril model of Lewy body disorders. Experimental Neurology, 299(A), 172-196. https://doi.org/10.1016/j.expneurol.2017.10.017

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Negin Nouraei and Daniel M. Mason contributed equally to this publication.

Rehana Leak is the corresponding author at 407 Mellon Hall, 600 Forbes Ave, Duquesne University, Pittsburgh, PA 15282, United States.

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School of Pharmacy

Abstract

Lewy body disorders are characterized by the emergence of α-synucleinopathy in many parts of the central and peripheral nervous systems, including in the telencephalon. Dense α-synuclein+ pathology appears in regio inferior of the hippocampus in both Parkinson’'s disease and dementia with Lewy bodies and may disturb cognitive function. The preformed α-synuclein fibril model of Parkinson’'s disease is growing in use, given its potential for seeding the self-propagating spread of α-synucleinopathy throughout the mammalian brain. Although it is often assumed that the spread occurs through neuroanatomical connections, this is generally not examined vis-à-vis the uptake and transport of tract-tracers infused at precisely the same stereotaxic coordinates. As the neuronal connections of the hippocampus are historically well defined, we examined the first-order spread of α- synucleinopathy three months following fibril infusions centered in the mouse regio inferior (CA2 + CA3), and contrasted this to retrograde and anterograde transport of the established tract-tracers FluoroGold and biotinylated dextran amines (BDA). Massive hippocampal α-synucleinopathy was insufficient to elicit memory deficits or loss of cells and synaptic markers in this model of early disease processes. However, dense α-synuclein+ inclusions in the fascia dentata were negatively correlated with memory capacity. A modest compensatory increase in synaptophysin was evident in the stratum radiatum of cornu Ammonis in fibril-infused animals, and synaptophysin expression correlated inversely with memory function in fibril but not PBS-infused mice. No changes in synapsin I/II expression were observed. The spread of α-synucleinopathy was somewhat, but not entirely consistent with FluoroGold and BDA axonal transport, suggesting that variables other than innervation density also contribute to the materialization of α-synucleinopathy. For example, layer II entorhinal neurons of the perforant pathway exhibited somal α-synuclein+ inclusions as well as retrogradely labeled FluoroGold+ somata. However, some afferent brain regions displayed dense retrograde FluoroGold label and no α-synuclein+ inclusions (e.g. medial septum/diagonal band), supporting the selective vulnerability hypothesis. The pattern of inclusions on the contralateral side was consistent with specific spread through commissural connections (e.g. stratum pyramidale of CA3), but again, not all commissural projections exhibited α-synucleinopathy (e.g. hilar mossy cells). The topographical extent of inclusions is displayed here in high-resolution images that afford viewers a rich opportunity to dissect the potential spread of pathology through neural circuitry. Finally, the results of this expository study were leveraged to highlight the challenges and limitations of working with preformed α-synuclein fibrils.

FIG.2 syn PBS bilateral box plots.ai (728881 kB)
FIG.2 syn PBS bilateral box plots.tif (123316 kB)
Fig. 2 α-synuclein fibril infusions lead to the emergence of dense inclusions in the hippocampal formation. CD1 mice were bilaterally infused with phosphate-buffered saline (1 μL PBS) or an equal volume of α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, sagittal brain sections were collected and stained with antibodies against the neuronal nuclear marker NeuN and pathologically phosphorylated α-synuclein (mouse anti-pSer129). Stitched images of immunostained sections at three sagittal levels of the hippocampal formation in one PBS-infused mouse and one fibril-infused mouse are displayed in A–C. Note that there may be slight imperfections at the boundaries of the computerized stitched images. The PBS-infused case is included to show the extent of background staining with the phospho-Ser129 antibody. The disruption of tissue in the overlying cortex reveals the path of the needle in panel A or B (white arrows). The adjacent NeuN and pSer129 images are from the same section viewed in different fluorescent channels; therefore, the anatomical labels are only placed on the NeuN image. The boxplots in panel D show a dramatic increase in the number of pSer129+ inclusions in Ammon’'s horn (CA1, CA2, and CA3), the stratum granulosum of the dentate gyrus (GrDG), the molecular layer of the dentate gyrus (MoDG), the presubiculum (PrS), and the posteromedial cortical amygdala (PMCo). The units shown on the Y axes are raw, unnormalized counts. **p ≤ 0.01, ***p ≤ 0.001 for PBS versus fibrils; two-tailed Mann-Whitney U test. n = 5 – –8 mice per group. For abbreviations, please consult Table 3.

FIG.4 synaptophysin synapsin.ai (6904 kB)
FIG.4 synaptophysin synapsin.tif (8663 kB)
Fig. 4 α-synuclein fibril infusions lead to a modest increase in synaptophysin levels in Ammon’'s horn. CD1 mice were bilaterally infused with phosphate-buffered saline (1 μL PBS) or an equal volume of α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, sagittal brain sections were collected and stained with antibodies against synaptophysin (A) or synapsin I/II (B). Staining was visualized on a low-resolution, ultrasensitive Odyssey Imager and a blinded observer traced the boundaries of CA2/CA3 or the dentate gyrus. The arbitrary units (a.u.) shown on the Y axes are raw, unnormalized fluorescence levels for protein content or pixel values for area. Representative low-resolution images are illustrated in C. Graphed are the mean + SEM. n = 8 – –10 mice per group. **p ≤ 0.05, **p ≤ 0.01 for PBS versus fibrils; two-tailed t-test. Rad = stratum radiatum; Pyr = stratum pyramidale.

FIG.5 colocalization rb ms pSer129 (2).ai (119436 kB)
FIG.5 colocalization rb ms pSer129 (2).tif (94621 kB)
Fig. 5 α-synuclein fibril infusions lead to the formation of ubiquitin and Thioflavin S-positive inclusions. CD1 mice were infused with α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, sagittal brain sections were collected and stained with mouse monoclonal and rabbit polyclonal antibodies against pathologically phosphorylated α-synuclein (pSer129) and guinea pig antibodies against NeuN (A). Nuclei were labeled with the Hoechst reagent. Note that the structures labeled in green in the merged image actually emit both red and green fluorescence, but that the greater green fluorescence intensity overwhelms the red label and the colocalization therefore does not appear yellow. (B) Quadruple staining with the Hoechst reagent and antibodies against pSer129, NeuN, and ubiquitin. (C–D) Sagittal sections from two representative vehicle-infused and two fibril-infused animals are shown after staining with Thioflavin S and the nuclear marker Hoechst. Note the dense Thioflavin staining in CA3 but not the fascia dentata. High magnification views of somal and neuritic Thioflavin S-stained inclusions in fibril-infused mice are shown in panel E. For abbreviations, please consult Table 3.

FIG.6 CA2 CA3 injxn pSer129 AI schemas only.ai (1064 kB)
FIG.6 CA2 CA3 injxn pSer129 AI schemas only.tif (10027 kB)
Fig. 6 Schematic map of α-synucleinopathy transmission through the brain. CD1 mice were infused with α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, sagittal brain sections were collected and stained with antibodies against pathologically phosphorylated α-synuclein (mouse anti-pSer129) and NeuN. Nuclei were stained with the Hoechst reagent. Schematized somal (red dots) and neuritic (red flourishes) pSer129+ inclusions were drawn on top of high-quality stitched images of whole brain sections. Only the most obvious anatomical boundaries were traced, after consulting the NeuN and Hoechst labeling. Two representative cases are shown in panels A and B and the case in panel C is shown because it harbored unusually dense pathology, perhaps from diffusion into the overlying cortex. The path of the needle in all three cases is illustrated by a black arrow. For abbreviations, please consult Table 3.

FIG.7 contra HP pSer129 Hoe NeuN merged.ai (121459 kB)
FIG.7 contra HP pSer129 Hoe NeuN merged.tif (119794 kB)
Fig. 7 α-synucleinopathy transmission to regions connected with the hippocampus. CD1 mice were infused with α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, coronal brain sections were collected and stained with antibodies against pathologically phosphorylated α-synuclein (mouse anti-pSer129). Nuclei were stained with the Hoechst reagent. Moderate to dense inclusions were found in the nucleus accumbens core (A–B) and the cortical amygdala (E–F). Small numbers of inclusions were found in the dorsal and ventral divisions of the lateral septum (C–D), the posterior hypothalamus (G–H), and the locus coeruleus and surrounding tegmentum (I–J). (K) High-magnification images of individual inclusions in various brain regions were captured with a 100 × oil objective. (L) The pattern of contralateral pSer129, NeuN, and Hoechst staining in coronal sections of the hippocampus in unilateral vehicle and fibril-infused animals is illustrated with images and schematics. Note that the majority of somal inclusions are in the stratum pyramidale of contralateral CA3. The PBS-infused case is included to show the extent of contralateral background staining with the phospho-Ser129 antibody. For abbreviations, please consult Table 3.

Fig.8 16-394 coronal pSer129 Rb and Ms (3).ai (362489 kB)
Fig.8 16-394 coronal pSer129 Rb and Ms (3).tif (375452 kB)
Fig. 8 α-synucleinopathy transmission through the ipsilateral and contralateral hemispheres, as shown in coronal sections. CD1 mice were infused with α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampal formation. Three months later, coronal brain sections were collected and co-stained with rabbit and mouse antibodies against pathologically phosphorylated α-synuclein (pSer129) and the nuclear marker Hoechst. Three coronal levels of the hippocampus are shown in this figure. The infused (right) hemisphere is on the left side of the images and a white arrow in the middle panel shows the path of the needle, with some disturbance in the tissue in the dorsal hippocampus and overlying cortex. The circular fiducial marks were made in the cortex and mesencephalon on the side opposite to the infusion. This infusion was centered more caudally than intended and exhibited some evidence of fibril diffusion up the needle track (see the inclusions in the cortex below the arrow indicating the needle path). The mouse monoclonal antibody elicited denser labeling of inclusions than the rabbit polyclonal. Note that there may be slight imperfections at the boundaries of the computerized stitched images. For abbreviations, please consult Table 3.

FIG.9 FluoroGold HP.ai (81342 kB)
FIG.9 FluoroGold HP.tif (161352 kB)
Fig. 9 Retrograde transport of the tract-tracer FluoroGold from the septal pole of the hippocampus. CD1 mice were infused with the retrograde tract-tracer FluoroGold in regio inferior (CA2 + CA3) of the dorsal hippocampus. One week later, sagittal brain sections were collected and nuclei were stained with the infrared marker DRAQ5 (purple). FluoroGold labeling was visualized in the UV channel. The center of the infusion site is shown in A–B. Dense retrograde labeling in the superficial layers of the caudomedial entorhinal cortex is shown in C–D. Retrograde label in the medial septum and ventral diagonal band of Broca is shown in E–F. Retrograde label in the lateral portions of the amygdalopiriform transition area is shown in G–H. (I–L) Commissural projection neurons in the contralateral hemisphere in two FluoroGoldinfused mice. Panels I–J are from a case with the infusion centered in the dentate gyrus and CA3, leading to contralateral retrograde label of hilar mossy cells in the polymorphic layer and CA3 pyramidal neurons. Panels K–L are from a case with the infusion centered in CA3 without dentate involvement, resulting in homotopic label in contralateral CA3 but not in hilar mossy cells.

Fig.10 BDA injection site montage HP.ai (559115 kB)
Fig.10 BDA injection site montage HP.tif (125577 kB)
Fig. 10 Injections of 10 kDa biotinylated dextran amines (BDA) in the septal pole of the hippocampus. CD1 mice were infused with the tract-tracer BDA (10 kDa) in regio inferior (CA2 + CA3) of the hippocampus. One week later, sagittal brain sections were collected and nuclei were stained with the Hoechst reagent (A, C, E, G, I). BDA was visualized with a streptavidin-conjugated fluorophore (B, D, F, H, J). Four separate animals are shown (A–B, C–F, G–H, and I–J) in order to illustrate the range of tracer diffusion. The case in panels A-B is centered in CA1 and CA2, the case in panels C–F is centered in CA2/CA3, the case in panels G–H is centered in CA1 and the dentate gyrus with diffusion into the thalamus, and the case in panels I–J is centered in CA3. The case in panels C–F is shown at two distinct sagittal levels to illustrate the topography of the retrograde label in the granule cell layer of the dentate gyrus (northeast arrow in panel F). The northwest arrow in panel F points to retrograde label in the parasubiculum and the arrow in panel J points to retrograde label in deeper layers of the entorhinal cortex.

Fig.11 BDA 3 KDa montage (2).ai (113271 kB)
Fig.11 BDA 3 KDa montage (2).tif (104335 kB)
Fig. 11 Anterograde and retrograde transport of 3 kDa biotinylated dextran amines (BDA) from the septal pole of the hippocampus. CD1 mice were infused with the tract-tracer BDA (3 kDa) in regio inferior (CA2 + CA3) of the hippocampus. Ten days later, sagittal brain sections were collected and nuclei were stained with the Hoechst reagent (A, C, E, G, I, K, M). BDA was visualized with a streptavidin-conjugated fluorophore (B, D, F, H, J, L, N). All brain sections shown in this figure are from one instructive case with diffusion into the optic tract of the underlying thalamus. The path of the needle is displayed with a white arrow in panel D. Although the injection was centered in CA2 + CA3, diffusion into the optic tract led to dense anterograde label of retinal projections to the superior colliculus and dense retrograde label of the optic chiasm in panel B (see two white arrows). This panel also shows anterograde label in the thalamus and retrograde label in the ventromedial hypothalamus. Dense anterograde label was observed in deep layers of the caudomedial entorhinal cortex in panel F, in addition to moderate retrograde label in superficial layers (white arrow).

FIG.S1 pSer129 co-injection photos (2).ai (107374 kB)
FIG.S1 pSer129 co-injection photos (2).tif (141552 kB)
Fig. S1 α-synucleinopathy transmission through the brain. CD1 mice were infused with α-synuclein fibrils (5 μg) into regio inferior (CA2 + CA3) of the hippocampus. Three months later, sagittal brain sections were collected and stained with antibodies against pathologically phosphorylated α-synuclein (mouse anti-pSer129; red) and the neuronal nuclear marker NeuN (purple). Adjacent panels are from the same brain sections, visualized in different fluorescent channels; therefore, labels are only placed on the NeuN image. Sections illustrated in panels A–H correspond to the brain illustrated schematically in Fig. 6A. Sections illustrated in panels I–P correspond to the brain illustrated in schematically in Fig. 6B. Sections illustrated in panels Q-V correspond to the brain illustrated in schematically in Fig. 6C. The scale bar in panel D also applies to panels E–H. For abbreviations, please consult Table 3.

FIG.S2 10x PoDG montage (2).psd (74786 kB)
FIG.S2 10x PoDG montage (2).tif (29549 kB)
Fig. S2 High-resolution image of specific layered pattern of inclusions in the hippocampal formation. A coronal section of the caudal hippocampus in a fibril-infused mouse is displayed in this figure. These sections were stained with mouse and rabbit pSer129 antibodies as well as the Hoechst reagent. Note the layered pattern of inclusion formation. For abbreviations, please consult Table 3.

FIG.S3 BDA 10kDa.ai (36189 kB)
FIG.S3 BDA 10kDa.tif (78798 kB)
Fig. S3 High-magnification images and schematic maps of anterograde and retrograde transport of 10 KDa biotinylated dextran amines (BDA) from the septal pole of the hippocampus. CD1 mice were infused with the tract-tracer BDA (10 kDa) in regioinferior (CA2 + CA3) of the hippocampus. One week later, sagittal brain sections were collected and nuclei were stained with the Hoechst reagent (blue). BDA was visualized with a streptavidin-conjugated fluorophore (green). (A–B) Dense anterograde and moderate retrograde label in the deep and superficial layers of the caudomedial entorhinal cortex, respectively. (C–E) Dense anterograde label in the molecular layer of the dentate gyrus and the dorsal lateral septum (white arrows). A representative 10 kDa BDA case is shown schematically in panel F. Higher magnification views of the lateral septal labeling is shown in panels G–J. One retrogradely labeled neuron is evident in panel J. For abbreviations, please consult Table 3.

FIG.S4 thal ctx injections.psd (40963 kB)
FIG.S4 thal ctx injections.tif (16419 kB)
Fig. S4 Control injections into the thalamus and cortex. CD1 mice were infused with α-synuclein fibrils (5 μg) into the cortex and thalamus. Three months later, coronal brain sections were collected and stained with rabbit (red) and mouse (purple) antibodies against pSer129. Nuclei were stained with the Hoechst reagent. (A–C) Injections in the thalamus led to sparse inclusions. Shown is a case with the densest pathology in the ventral posterior nucleus of the thalamus. All other cases exhibited far less pathology. (D–F) Following thalamic injections, some animals exhibited pSer129 in the claustrum. (G–I) Following injections in the cortex, the deeper layers of the cortex contained pSer129 label. The case shown here had the densest pathology of all cortex injections. For abbreviations, please consult Table 3.