Distinct involvement of the cranial and spinal nerves in progressive supranuclear palsy

Abstract

The most frequent neurodegenerative proteinopathies include diseases with deposition of misfolded tau or α-synuclein in the brain. Pathological protein aggregates in the PNS are well-recognized in α-synucleinopathies and have recently attracted attention as a diagnostic biomarker. However, there is a paucity of observations in tauopathies. To characterize the involvement of the PNS in tauopathies, we investigated tau pathology in cranial and spinal nerves (PNS-tau) in 54 tauopathy cases [progressive supranuclear palsy (PSP), n = 15; Alzheimer's disease (AD), n = 18; chronic traumatic encephalopathy (CTE), n = 5; and corticobasal degeneration (CBD), n = 6; Pick's disease, n = 9; limbic-predominant neuronal inclusion body 4-repeat tauopathy (LNT), n = 1] using immunohistochemistry, Gallyas silver staining, biochemistry, and seeding assays. Most PSP cases revealed phosphorylated and 4-repeat tau immunoreactive tau deposits in the PNS as follows: (number of tau-positive cases/available cases) cranial nerves III: 7/8 (88%); IX/X: 10/11 (91%); and XII: 6/6 (100%); anterior spinal roots: 10/10 (100%). The tau-positive inclusions in PSP often showed structures with fibrillary (neurofibrillary tangle-like) morphology in the axon that were also recognized with Gallyas silver staining. CBD cases rarely showed fine granular non-argyrophilic tau deposits. In contrast, tau pathology in the PNS was not evident in AD, CTE and Pick's disease cases. The single LNT case also showed tau pathology in the PNS. In PSP, the severity of PNS-tau involvement correlated with that of the corresponding nuclei, although, occasionally, p-tau deposits were present in the cranial nerves but not in the related brainstem nuclei. Not surprisingly, most of the PSP cases presented with eye movement disorder and bulbar symptoms, and some cases also showed lower-motor neuron signs. Using tau biosensor cells, for the first time we demonstrated seeding capacity of tau in the PNS. In conclusion, prominent PNS-tau distinguishes PSP from other tauopathies. The morphological differences of PNS-tau between PSP and CBD suggest that the tau pathology in PNS could reflect that in the central nervous system. The high frequency and early presence of tau lesions in PSP suggest that PNS-tau may have clinical and biomarker relevance.

Keywords: cranial nerve; peripheral nervous system; progressive supranuclear palsy; spinal nerve; tauopathy.

Figure 1

Representative photomicrographs of tau pathology in the PNS in progressive supranuclear palsy. (A–D) In the midbrain (A), phosphorylated tau deposits are evident in the oculomotor nerves: III, intra-medullary fibres (B: high magnification image of black box ‘b’ in A) and extra-medullary fibres [C: high magnification image of black box ‘c’ in A; arrow: phosphorylated-tau (p-tau) deposits; and D]. (E–H) In the medulla oblongata (E), tau accumulates in the glossopharyngeal/vagus nerves: IX/X, cross sections (F: high magnification image of black box ‘f’ in E; arrows: p-tau deposits; enlarged image in the top right corner) and sagittal sections (G). Occasionally, large inclusions with fibrillary morphology are also seen (H). (I–M) There are massive amounts of p-tau deposits in the spinal anterior roots [progressive supranuclear palsy (PSP) Case 1; J: high magnification image of black box ‘j’ in I; and K: high magnification image of ‘J’, arrow: p-tau deposits primarily located externally) and small amounts of p-tau deposits in the posterior roots (L, arrow; PSP Case 3) at the cervical cord level. (M) Tau pathology is also evident in the anterior horn of the cervical cord, PSP Case 1 (arrow: neurofibrillary tangle-like inclusions; and arrowhead: pre-tangle-like inclusions). (A–C) PSP Case 15, (D, G–K and M) PSP Case 1, (E and F) PSP Case 11 and (L) PSP Case 3. (A, E and I) Luxol fast blue/haematoxylin and eosin (LFB/H&E) and (B–D, F–H and J–M) AT8. Scale bars in A = 2 mm, B and M = 50 μm, C, D, F–H, J and L = 20 μm, E = 1 mm, I = 200 μm and K and enlarged image of the top right corner in F = 10 μm.

Figure 2

Staining profiles of PNS-tau in progressive supranuclear palsy (PSP) and comparing morphological features of tau-positive inclusions in PSP and corticobasal degeneration (CBD). (A–G) Representative photomicrographs of staining profiles of the PNS-tau in PSP. The tau-positive inclusions in the PNS are detected with 4-repeat tau specific antibody (RD4) (A) but not with 3-repeat tau-specific antibody (RD3) (B). The tau-positive inclusions are also recognized with several phosphorylated tau antibodies [C(i and ii): AT180 and D: pThr217] other than AT8 and also Alz-50, which is for misfolded tau (E). The inclusions in PSP often show large structures with fibrillary morphology (A and E), which are stained with Gallyas–Braak silver staining (G-B) [F(i and ii)]. Some inclusions also show p62-immunoreactivity [G(i and ii)]. (H–K) Representative photomicrographs of tau pathology in the PNS and related nuclei in CBD Case 3, namely anterior horn (H), spinal anterior roots (I and J: high magnification image of black box ‘j’ in I) in the cervical cord and IX/X nerve (K). Although this CBD case shows massive tau pathology in the anterior horn of the cervical cord (H, arrowhead: pre-tangle-like inclusions), there were only a few fine granular tau deposits in the spinal anterior roots (I and J). (K) A small number of granular tau deposits is present in the IX/X nerve. (A) RD4, (B) RD3, (C) AT180, (D) pThr217, (E) Alz-50, (F) G-B, (G) p62 and (H–K) AT8. Scale bars in A–C(i and ii), E, F(i and ii), G(i and ii), J and K = 10 μm, D = 20 μm, H = 100 μm and I = 50 μm.

Figure 3

Double-labelled immunofluorescence of phosphorylated-tau (p-tau) and neurofilament/S100β: the morphological features and localization sites of p-tau deposits in the peripheral nerve fibres. (A) In cross sections, phosphorylated tau deposits are primarily localized within the neuronal fibres (i)/axons (ii). (B) Some of the nerve fibres are filled with large tau inclusions showing fibrillary morphology (i–iii). (C) A part of the large tau-positive inclusions appear to accumulate in the lateral portion of nerve fibres, suggesting the possibility that the deposits are partly located within myelin. However, p-tau and S100β are not completely co-localized, and p-tau deposits merely appear to overlap on the myelin [C(i): cross and C(ii): sagittal section]. (D) Sagittal section of D-IF: p-tau and p-NF. In support of this, p-tau deposits with the outer portion are present within the axons (p-NF). In addition, the axons detected with p-NF seem to be bulged due to the large tau-positive inclusions. [A(i) and B(i)] NF (green) and pThr217 (red), [A(ii), B(ii and iii) and D] SMI31 (green) and pThr217 (red) and (C) AT8 (green) and S100β (red). Scale bars in A–D = 10 μm.

Figure 4

Tau immunoblot analysis and in vitro tau seeding assay. (A) Tau immunoblot, total tau (HT7 clone), reveals the presence of tau in all four samples. (B–E) In vitro tau biosensor cells demonstrate that tau deposits (green) in the primary motor cortex (D and E) and cervical spinal nerve (C, enlarged image in the bottom left corner) samples have seeding capacity. (B and D) PSP Case 8 (PSP 8) and (C and E) PSP Case 10 (PSP 10). PMC = primary motor cortex; SpN = spinal nerves. Scale bars in B–E = 100 μm and C, enlarged image in the bottom left corner = 10 μm.