Myelinated Ependymal tanycytes in human and mouse hippocampus project into the hippocampal stratum pyramidale and give rise to waste-internalizing receptacles.

(A-B) The alveus of human hippocampus contains numerous Luxol H&E-stained ependymal tanycytes that send vast numbers of slender myelinated processes (white arrow in B) into the stratum pyramidale of the hippocampal formation. Numerous electron-lucent ‘swell-bodies’ with associated nucleus-like tanysomes are forming along individual myelinated processes (black arrowheads in B). (C, D) Aquaporin4-immunolabeling of human alveus shows strong immunoreactivity of tanycyte processes. Higher magnification (D) shows Hoechst-blue stained nucleus-like structures in close association with tanycytes (black double arrowheads in D). Circular, immunoreactive swellings can be observed along the tanycyte processes (black arrowheads). (E-G) Luxol-blue stained human tanycyte processes (white arrows) project into the stratum pyramidale where they form translucent swell-bodies (black arrowheads) with associated nucleus like tanysomes (black double arrowheads). Please note the close association of swell-bodies with neuronal somata (asterisks). (H) Tanycyte (white arrow) with associated translucent swell body (black arrowhead) with tanysome (black double arrowhead) with emanating waste receptacles (white double arrowhead). Swelling ring structures (black arrows) that line the inside of swelling tanycyte processes are visible in Luxol H&E-stained and Aβ-immunolabeled (black arrow in inset) tanycyte processes. (I-K) AQP4 (red) and myelin (green) immunolabeling of mouse hippocampus shows double labeled tanycyte processes that contain myelin-immunoreactive circular profiles that line the inner lumina of the cell processes (white arrows). Black arrowhead: AQP4-immunolabled swell body. (L-N) Tanycyte processes (white arrows) with associated swell-bodies (black arrowheads) in human stratum pyramidale. White double arrowheads: forming receptacles; Black double arrowheads: Tanysomes. Inset in M: Forming tanysomes in the alveus. (O-T) Structural characteristics of swell bodies, associated tanysomes (black double arrowheads) and receptacles (white double arrowheads) in human hippocampus. Both light and ultrastructural images show the electron lucent nature of the swell bodies. Each swell body contains between one and 6 tanysomes that are stained for nuclear stain in Luxol H&E-stained preparations and appear associated with myelinated tanycyte processes (white arrows). Tanysomes give rise to toroids and receptacles some of which appear electron-dense at the ultrastructural level (white double arrowheads in T). Please note the myelinated tanycyte profiles that line the circumference of swell bodies (small white arrows in O, P, T). (U-W) AQP4/Myelin immunolabeled swell body in mouse hippocampus shows the double-labeled nature of myelinated AQP4-immunoreactive tanycyte profiles (white arrow) around the periphery of swell bodies. (X) Typical structured appearance of a glial nucleus (asterisk) and surrounding cytoplasm (white arrowhead). Scale bars: A: 500 µm, B:10 µm, C: 20 µm; D: 5 µm; E-G: 10 µm, H: 5 µm, Inset 3 µm; I-N: 10 µm; M: 20 µm; N: 10 µm; O: 5 µm, (Inset) 2 µm; P: 5 µm Q 2 µm R: 5 µm; S: 5 µm; T: 2 µm; U-W: 5 µm; X: 2 µm.

Tanysomes in human and mouse hippocampus give rise to toroids and receptacles that are immunoreactive for Aβ, hyperphosphorylated tau protein, myelin, AQP4 and α-tubulin.

(A-O) Human hippocampal tanysomes within swell-bodies give rise to a network of membranous receptacles and toroids (white double arrowheads). The physical attachment between tanysomes (black double arrowheads) and tanycyte processes (white arrows) and receptacles (grey arrows) is clearly visible. Nuclear stain is observed in both tanysomes and emanating toroid-shaped ring structures (F, G, H). The tanysome-derived membrane network in human brain is immunoreactive for tau-protein (E, L), Aβ (G, H, I, K, M, N) and α-tubulin (O). (P-R) Immunolabeling of mouse hippocampus for anti-AQP4 (red) and myelin (green) demonstrates that toroids that emerge from tanysomes are immunoreactive for both epitopes. (S-U) Tanysome-derived receptacles in preparations immunolabeled for anti-AQP4 (S), anti-Aβ (T) and stained for Luxol H&E (U) consistently demonstrate their physical association with tanysomes (grey arrows). Scale bars: A-U: 5 µm.

Tanysome-derived waste receptacles in human hippocampus project into neuronal somata.

(A) Tanysome-derived (black double arrow) waste receptacles are immuno-positive for aquaporin-4 (white double arrowhead). (B-G) Luxol H&E-stained preparations show the characteristically blue stained tanysome-derived waste receptacles that differentiate in swell-bodies (panel B) project into pyramidal cell somata. Black double arrowheads: Tanysomes; White double arrowheads: Waste receptacles; Black arrowheads: Outer margins of swell-bodies; N: Neuronal somata. (H-J) Ultrastructural depiction of healthy and degenerating neuronal somata demonstrates the abundance of swelling waste receptacles and gradual depletion of cytoplasmic areas in degenerating somata (H, J) compared to the much sparser number of waste receptacles in a healthy neuron that shows an intact cytoplasmic structure (I). Please note the waste accumulation associated with a waste receptacle strand that emanates from a myelinated cell profile (arrows in H) consistent with strands of waste receptacles observed at the light microscopic level (C-G). Scale bars: A-G: 5 µm; H-J: 2 µm.

Evidence for aquaporin-4-dependent Cy3 goat anti-rabbit antibody internalization into living mouse hippocampal ependymal tanycytes.

(A-C) Living ependymal tanycytes that were exposed to Cy3 goat anti rabbit antibody appear fluorescent after 30 min exposure (white arrowheads in A). Immunolabeling against goat-protein using an Alexa 488-coupled donkey-anti-goat antibody demonstrates co-localization of both Cy3 and Alexa 488 fluorochromes (white arrowheads in B, C) indicative that the red fluorescence within tanycytes originates from internalized Cy3 goat anti rabbit antibody. (D) Ependymal tanycytes in mouse alveus show red fluorescence after 30 min exposure of living brain tissue to Cy3 goat anti-rabbit antibody (white arrowheads). A fine network of fluorescing processes (grey arrowhead) is visible in the stratum pyramidale. Insets: Canal structures each containing two fluorescing channels (arrows) are frequently observed projecting into the adjacent ventricle. (E, F) Normalized intensity of internalized Cy3 fluorochrome comparing control (0; E) and AQP4-blocked (60 µM TGN-020; F) conditions. Control cells demonstrate a higher pixel intensity, especially towards the alveus. (G) Log10 mean cellular object intensity is significantly larger in control (0 µM;-1.888) compared to AQP4-blocked (60 µM TGN-020; −2.278; t(928) = 32.666, p = 2.44 × 10−156, unpaired two-tailed t-test). Inset in E: Fluorescent canal structures extending into the ventricle (white arrows). (H) Schematic depiction of the proposed waste internalization demonstrated here. We postulate that living tanycytes internalize surrounding fluorochromes via extracellular waste receptacles that are formed within swell-bodies explaining the observed fluorescence in ependymal tanycytes. To visualize cells, Hoechst blue nuclear stain was applied after fixation of the brains. (I) Single 3-µm optical confocal section of mouse brain with internalized fluorochromes. Note the strong resemblance of brightly fluorescent tanysome-like areas (1, 3, 5, 6) with toroid- and receptacle-forming human tanysomes (2, 4) and AQP4/myelin-doublelabeled mouse tanysomes (7-9). Scale bars: A-C: 10 µm, D: 50 µm; E, F: 20 µm; H: not drawn to scale; I: 5 µm.

RNA-scope gene expression within tanycytes and associated swell-bodies of human hippocampus in both AD-affected and AD-unaffected tissue.

(A) Electron micrograph of a tanycyte soma (white arrowhead) in the alveus of AD-affected tissue shows myelinated tanycyte processes projecting from the soma (white arrows). (B, C) RNA-scope visualization of RNA expression for glial-fibrillary acid protein (GFAP; green) and aquaporin-4 (AQP4; red) demonstrate the expression of both RNAs in the alveus (white arrowheads). Please note the abundance of GFAP within tanycyte processes indicative of RNA-transport (green arrows). (D, E) AQP4-RNA expression in the stratum pyramidale counter-stained with DAPI nuclear stain shows brightly fluorescing tanysomes (black double arrowheads) with emerging waste receptacles (white double arrowheads). Please note the absence of staining in the astrocyte-like cell soma (grey arrows). (F) Luxol H&E stained tanysome (black arrowhead) with emerging receptacle-forming toroid (white double arrowhead) strongly resembles the AQP4-expressing tanysome indicated by the asterisk in D, E. (G, H) AQP4-expressing waste receptacles (white double arrowheads) that emerge from tanysomes (black double arrowheads). Cell somata resembling a neuron (white arrow) and adjacent astrocyte (grey arrow) are void of AQP4-RNA expression. (I) Tanysome (black double arrowhead) with emerging anti Aβ-stained toroid (white double arrowheads) strongly resembles the AQP4-RNA expressing tanysome indicated by the asterisks in G, H. (J-M) Receptacle-forming (white double arrowheads) tanysome (black double arrowheads) show co-expression for GFAP (green, white double arrowheads) and AQP4 (red). (N) Anti-tau-stained tanysome (black double arrowhead) with associated receptacle-forming toroid (white double arrowheads) strongly resemble the tanysome shown in J-M. (O-P) AQP4-RNA expression is restricted to receptacle-forming tanysomes (white and black double arrowheads) that are in close contact to an unstained neuronal soma (white arrow) and an unstained astrocyte-like soma (grey arrow). (Q) Luxol H&E-stained pyramidal cell soma (white arrow) shows the consistency of the RNA-scope labeling in O, P whereby receptacle forming tanysomes (white and black double arrowheads) are adjacent to neuronal soma. Scale Bars: A: 2 µm; B, C: 5 µm; D-H: 10 µm; I: 5 µm; J-Q: 10 µm.

Amyloid β-related gene expression in AD-affected human hippocampus.

(A-D, F-J, L-P) Ependymal tanycytes in the ventricular lining show RNA-co-expression for Presenilin-1 (Pres-1, magenta), Amyloid Precursor Protein (APP, green) and Aquaporin 4 (AQP4, red). Higher magnification (F-J, L-P) shows that gene expression is associated with tanysome-derived toroids and receptacles (DAPI nuclear stain). (E) Tanysome-associated waste receptacles are strongly Aβ-immunoreactive (white double arrowheads) consistent with observed Pres1 and APP gene expression shown in F-J. (K) Anti-tau immunoreactive tanysome-associated toroid. (Q, R) Anti Aβ-immunoreactive toroid (white double arrowhead) with emerging waste receptacles (black and grey arrows). Grey arrows indicate the area shown at higher zoom in (R). (S-U) Electron micrographs of forming waste receptacles (white double arrowheads) in swell-bodies that emerge from myelinated cell profiles (black arrowheads). (V-Ai) Tanysome-associated waste receptacles show immunolabeling for Pres1 (V, W), APP (W, X), Tau protein (Y) and Caspase 2 (Z). The appearance of the immunolabeled receptacles is consistent with the ultrastructural appearance of waste-containing receptacles (Ai). Scale Bars: A-D: 20 μm; E-R: 5 μm; S: T: U: 2 μm; V-Z: 5 μm; Ai: 500 nm.

Tanysome-derived waste receptacles stain for Aβ, tau protein, and α-tubulin in AD-affected human brain tissue

(A-B) Alzheimer Disease unaffected brain tissue shows moderate immunolabeling for Aβ and tau protein in association with intraneuronal (A) and extracellular (B) waste receptacles. Generally, the signal for Aβ is stronger compared to the observed anti-tau immunolabeling. (C-Q) Comparison of Luxol H&E-stained tanysome-derived receptacles and unraveling Luxol blue-stained ring structures in AD-affected brain tissue (C, F, O) demonstrates their structural similarities to Aβ immunolabeled (D, E, G, I, J, K), Anti-tau immunoreactive (L, M, N, Q) and anti α-tubulin immunolabeled (H) preparations. Please note the similarities of unraveling myelin-derived cell profiles of varying sizes (O) with unraveling myelinated cell profiles at the ultrastructural level (P). These unraveling toroids are consistent with unraveling toroids observed in the anti-tau labeled preparations (L, M, N, Q) that have the characteristic appearance of ‘tau tangles’ (grey-white double arrowheads). Black double arrowheads: Tanysomes; White double arrowheads: Forming receptacles; Black arrowheads: outer margins of swell-bodies; N: Neuronal soma. Scale bars: A-O: 5 µm; P: 500 nm; Q: 3 µm.

Light- and electron-microscopic architecture of tanycyte-derived waste receptacles formed within swell bodies in the human brain.

(A, B) Schematic depiction of the proposed structures that form within swell-bodies (A) and can be observed to project into neuronal somata (B). Swelling toroids (1) that are connected to an adjacent tanysome (2) via a slender tanycyte process (5). The tanysome gives rise to myelinated toroids (3) from which strands of waste-internalizing receptacles emerge (4). The individual receptacle strands terminate into a distal toroid-shaped ring structure (8) that merge into distal tanycyte processes (7). (C-K) The above-described structures and their proposed Luxol H&E-stained (C, F), anti-tau immunolabeled (B, H, I) and anti-Aβ immunolabeled (G, J, K) equivalents in AD-affected hippocampus are depicted and labelled accordingly within each panel. The tanysome-derived toroids and associated receptacle strands (3, 4) appear electron dense at the ultrastructural level and form large numbers of receptacles (D, E). The emerging tubular canal structures resemble glial fibrillary acid protein (GFAP, black arrowhead in E). Waste receptacles can be seen emanating from toroid-shaped ring structures in Luxol H&E-stained preparations (white arrowhead in F). Immunolabeling for Aβ shows similar immunolabeled waste receptacles (G, J, K and inset). Higher zoom shows the association of individual receptacles with a fine network of fibrillary canals consistent with the postulated stabilization of these proposed waste-internalizing structures by Aβ (inset in K). 6: outer perimeter of swell body; Scale bars in light microscopic images: 5 µm; D: 500 nm; E: 150 nm. Schematic drawings not drawn to scale.

Hypertrophic tanycyte swelling in human AD-affected brain tissue.

(A-C) Tanycyte processes in the alveus (white arrows) in non-AD affected brain appear linear and project parallel to each other in both anti-tau labeled brain tissue (A-C) and Luxol H&E-stained preparations (inset in A). Tanysome (black double arrowheads) derived waste receptacles are sparse and appear either unlabeled or weakly labeled for tau protein (black arrows). (D-F) The alveus in AD-affected brain tissue appears spongiform with bulging tanycyte processes in both anti-tau (D-F) and Luxol H&E-stained preparations (inset in D). Numerous swelling anti-tau labeled waste receptacles (black arrows) emanate from associated tanysomes (back double arrowheads). (G, H) Similar hypertrophic swelling can be observed in tanycyte processes (white arrows in G compared to H) and intraneuronal tanycyte receptacles (white double arrowheads in G compared to H, higher zoom of receptacles in respective insets). (I-N) Swell bodies and associated tanycyte profiles in non-AD affected tissue appear comparatively small and intact (I) compared to those in AD-affected brain tissue (J-N). In the latter swell bodies, tanysome-derived waste receptacles and associated tanycytes show abnormal hypertrophy (J-L). Please note the accumulation of brown deposit within the hypertrophic tanycyte process, indicative of a blockage by cellular waste (red arrowhead in J). Tanysomes and associated waste receptacles are difficult to recognize in swell-bodies that show advanced swelling stages (K, L higher zoom that visualizes remaining waste receptacles). Hypertrophic tanycyte processes form numerous swelling circular structures in comparison to those in non-AD affected tissue (arrows in I compared to M, N, inset in N). (O, P) Differences in swelling patterns are also observed in Aβ immunolabeled tanycyte-derived toroids (arrows in O and P). (Q, R) Comparison between non-AD (Q) and AD-affected tissue (R) at the ultrastructural level demonstrates abnormal spongiform swelling that originates from bulging myelin-derived tanycyte protrusions, (white arrows in Q compared to R). Scale bars: A-P: 5 µm, Q, R: 2 µm.

Schematic depiction of proposed tanycyte-derived waste canal system in the human hippocampus of healthy (A) and AD-affected brain tissue.

(A) Neurons (blue) and glial cells (green) are contacted by myelinated aquaporin-4-expressing ependymal tanycytes (beige). The latter form both swelling toroids and waste receptacles (1-5) either directly from tanycyte protrusions [11] or swell-body-derived that internalize and remove cellular waste from the brain parenchyma likely via apical drainage canals (6-8) in a proposed AQP4-dependent manner. We propose that receptacle formation is likely dependent on: (i) Amyloid β-mediated stabilization to prevent the collapse of waste-internalizing receptacles during this process, and (ii) tau-protein for the gradual and time appropriate release of waste receptacles. (B) In AD-affected brain tissue, hypertrophic tanycyte abnormalities that may be caused by physical waste obstruction lead to swelling of the tanycytes affecting the brain parenchyma (9, 10), intracellular waste receptacles that lead to dense obstruction of affected neurons and glial cells (11, 12, 13), tanycyte processes (14), and intraneuronal tanysome-derived receptacles (15, 16, 17). Swelling of tanycytes in the alveus leads to excessive sprouting of tanycyte-derived waste receptacles that appear Aβ immunoreactive (18, 19). Similar swelling can be observed in tanycyte-associated swell-bodies (20) and toroids (21). Schematics are not drawn to scale.

Proposed AQP4-mediated uptake mechanism of cellular waste into tanysome-derived waste receptacles.

Swell-bodies differentiate myelin-derived AQP4-expressing receptacles that create a convective cytoplasmic flow toward the receptacles (light blue arrows) that flushes cellular debris particles toward the receptacles (red arrows). We postulate that receptacle-associated enzymes (e.g., Caspase 2) catabolize the debris prior to uptake into tanycytes. We propose that water influx into tanycytes may be calcium-mediated through the synergistic activation of TRPV4 cation channels. We postulate that swelling toroids that are formed along tanycytes may draw waste from the receptacles toward the toroids (dark blue arrows). We postulate that these canals may contain membrane-gated valves that prevent backflow. Schematic not drawn to scale.