Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems

Filaments made of residues 120-254 of transmembrane protein 106B (TMEM106B) form in an age-dependent manner and can be extracted from the brains of neurologically normal individuals and those of subjects with a variety of neurodegenerative diseases. TMEM106B filament formation requires cleavage at residue 120 of the 274 amino acid protein; at present, it is not known if residues 255-274 form the fuzzy coat of TMEM106B filaments. Here we show that a second cleavage appears likely, based on staining with an antibody raised against residues 263-274 of TMEM106B. We also show that besides the brain TMEM106B inclusions form in dorsal root ganglia and spinal cord, where they were mostly found in non-neuronal cells. We confirm that in the brain, inclusions were most abundant in astrocytes. No inclusions were detected in heart, liver, spleen or hilar lymph nodes. Based on their staining with luminescent conjugated oligothiophenes, we confirm that TMEM106B inclusions are amyloids. By in situ immunoelectron microscopy, TMEM106B assemblies were often found in structures resembling endosomes and lysosomes. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-024-01813-z.


Introduction
Transmembrane protein 106B (TMEM106B) was first linked to neurodegenerative diseases, when sequence variations in its gene were identified as a risk factor for frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), especially in individuals with granulin gene (GRN) mutations [42].TMEM106B has also been implicated in neurodegenerative diseases other than ALS/FTLD-TDP [16,31].Moreover, genome-wide association studies have suggested a role for TMEM106B in age-associated phenotypes in the cerebral cortex, independently of disease, with increased neuronal density in cases with the protective haplotype [27,34].Outside the nervous system, TMEM106B has been identified as a receptor for SARS-CoV-2 cell entry in an angiotensinconverting enzyme 2 (ACE2)-independent manner [5,37].
Electron cryo-microscopy (cryo-EM) has shown that amyloid filaments of TMEM106B can be found in human brains in an age-dependent manner [8,14,21,23,39].They can be extracted from the brains of neurologically normal subjects and of individuals with neurodegenerative diseases, including those with filamentous inclusions made of amyloid-beta, tau, alpha-synuclein or TDP-43.TMEM106B inclusions do not co-localise with other protein aggregates [39,49].
An increase in TMEM106B levels with age has been reported in carriers of the non-coding rs1990622 risk allele [19,26].The risk allele has also been associated with a higher number of TMEM106B aggregates [26,28].Conversely, the change from T185 to S185 (encoded by rs3173615) protects against FTLD-TDP [12,30].
TMEM106B is a single-pass, type II transmembrane protein that is predominantly found in late endosomes and lysosomes; some protein may also localise to the plasma membrane [5,16,31].TMEM106B is expressed ubiquitously, with highest levels in brain, heart, thyroid, testis and adrenal gland.In the brain, the expression of TMEM106B has been reported to be highest in neurons and oligodendrocytes and it has been implicated in myelination [51].TMEM106B comprises a cytoplasmic portion (residues 1-96), followed by a transmembrane region (residues 97-117) and an intraluminal part (residues 118-274); the latter adopts a compact fibronectin type III domain with a seven-blade beta-sandwich fold [5].The intraluminal portion carries glycosylation motifs at N145, N151, N164, N183 and N256 [25,28].
TMEM106B is cleaved physiologically by an unknown protease that releases its C-terminal domain into the lysosomal lumen [6].This generates a residual N-terminal fragment that is anchored to the lysosomal membrane.It is further cleaved by signal peptide peptidase-like 2A (SPPL2A) through intramembrane proteolysis, releasing an intracellular N-terminal domain into the cytosol and a small C-terminal domain into the lumen [32].
Release of the C-terminal domain of TMEM106B following cleavage at S120 is probably necessary for the formation of filaments, which extend from residues 120-254, suggesting that amino acids 255-274 are either unstructured or removed through a second cleavage.Here we show that such a cleavage appears likely, based on staining with an antibody specific for the C-terminus of TMEM106B (residues 263-274).
We also show that inclusions labelled by TMEM106B antibodies are fluorescent with amyloid dyes [4]; they were detected throughout central and peripheral nervous systems, where they were most abundant in glial cells.No inclusions were found in heart, liver, spleen or hilar lymph nodes of subjects with TMEM106B aggregates in the brain.By immunogold electron microscopy of cortical tissue sections TMEM106B filaments were located in vacuole-like structures and in structures resembling late endosomes and lysosomes.

Cases
Table 1 and Supplementary Table 1 summarise characteristics of the cases investigated in this study.The cases used in the main figures are listed in Table 1.The 113 cases studied included 41 neurologically normal controls, 7 sporadic cases of Alzheimer's disease (AD), 2 familial AD cases (one with PSEN1 and one with APP mutation), 15 cases of progressive supranuclear palsy (PSP), 6 cases of Pick's disease (PiD), 2 cases of argyrophilic grain disease (AGD), 5 cases of frontotemporal dementia and parkinsonism linked to chromosome 17 caused by a MAPT mutation (FTDP-17T), 10 Parkinson's disease (PD) cases (2 with GBA mutations, 8 sporadic), 2 cases of Parkinson's disease dementia (PDD) (with GBA mutations), 2 cases of Lewy body dementia (LBD) (one with a GBA mutation), 2 cases of frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), 3 cases of multiple sclerosis, 2 cases of Sanfilippo A syndrome, 3 cases of neuroserpin, and one case each of juvenile-onset synucleinopathy (JOS), Alper's disease, Down's syndrome, Huntington's disease, Friedreich's ataxia, motor neuron disease (with C9orf72 repeat expansion), vascular dementia, congophilic angiopathy, AD with atypical tauopathy, cerebellar degeneration, and dementia.In addition, we used brains from 7-month-old homozygous mice transgenic for human mutant P301S tau [1] and whole brains from 3-to 24-month-old control mice.

Immunohistochemistry
Single-labelling immunohistochemistry was carried out as described [39], with minor modifications.Specificity of the staining was established by antibody pre-adsorption (Supplementary Fig. 1).Following deparaffinization, formalin-fixed sections underwent heat-induced epitope retrieval (HIER) in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9) at 90 °C for 20 min.Other epitope retrieval methods tested included HIER in 10 mM citrate buffer (1.8 mM citric acid, 8.2 mM trisodium citrate, pH 6) at 90 °C for 20 min, and formic acid (87%) pre-treatment at room temperature (RT) for 10 min (Supplementary Fig. 1).This was followed by overnight incubation with primary antibody at 4 °C.Following washing in phosphate-buffered saline with 0.3% Triton X-100 (PBST), the sections were incubated with ImmPRESS-HRP polymer anti-rabbit detection antibody (Vector Laboratories) for 2 h at room temperature.Following washing with PBST, ImmPACT Vector SG substrate (peroxidase) was added to visualise the signal.Sections were counterstained with nuclear fast red (Vector Laboratories).For double-labelling immunohistochemistry, following completion of staining with a rabbit primary antibody, the sections were incubated overnight at 4 °C with the mouse primary antibody against the second antigen.Following washing in PBST, the sections were incubated with ImmPress-AP polymer anti-mouse detection antibody (Vector Laboratories) for 2 h at room temperature.Following washing, the second antigen was visualised with impact Vector Red substrate (alkaline phosphatase).Following dehydration, the sections were coverslipped with Entellan mounting medium (Merck).Images were acquired using a QImaging Retiga 2000 CCD camera mounted on an Olympus BX50 microscope.

Immunofluorescence and staining with amyloid dyes
Following incubation with primary antibodies overnight at 4 °C, the sections were washed in PBS and treated with TrueBlack lipofuscin autofluorescence quencher (Biotum) for 2 min at room temperature.Following washing in PBS, they were incubated with Alexa Fluor 568 and/or 647-conjugated secondary antibodies (Invitrogen, 1:250 in PBS) for 2 h at room temperature.Some sections were then labelled with either pentamer-formyl thiophene acetic acid (pFTAA, 3 µM), hepta-formyl thiophene acetic acid (hFTAA, 3 µM), HS-68 (3 µM) or Amytracker 540 (Ebba Biotech, 1 µM) in PBS or Thioflavin S (1% in distilled water) for 2 h at room temperature.Nuclei were counterstained with Hoechst dye (2.5 µg/ml in PBS, Sigma Fine Chemicals) for 15 min at room temperature.The sections were coverslipped with Fluoromount-G mounting medium (Southern Biotech).Images were captured on a Leica Stellaris 8 confocal microscope and processed using ImageJ.

Immunogold electron microscopy of tissue sections
We used cerebral cortex from a 90-year-old male with AD (Table 1) and abundant TMEM106B inclusions.Tissue had been stored in formalin and one mm 3 blocks were resected and fixed further in 2.5% glutaraldehyde with 2% paraformaldehyde overnight at 4 °C.Fixative was washed from the tissues with cacodylate buffer.Tissues were then treated with 1% osmium tetroxide and 1.5% potassium ferrocyanide in cacodylate for 1 h at room temperature.
Samples were dehydrated in a graded ethanol series, incubated in acetone for 30 min and embedded in EPON resin for 48 h at 60 °C.Ultrathin Sections (70 nm) were cut using a diamond knife and placed on 200 mesh nickel grids (EM Sciences FCF200-Ni).The grids were floated on blocking solution (0.1% BSA in PBS) for 1 h at room temperature, followed by incubation with the TMEM239 primary antibody diluted 1:100 in blocking solution, overnight at 4 °C.Excess primary antibody was washed from the grids with wash buffer (0.01% BSA-C in PBS).The grids were floated on 10 nm immunogold goat-antirabbit-secondary antibody (EM Sciences 25109) diluted 1:40 in blocking solution for 2 h at room temperature.Excess secondary antibody was removed with wash buffer, followed by distilled water.The sections were contrasted for one min with UranyLess (EM Sciences), washed with water, blotted and dried in a vacuum dessicator for 1 h before imaging.All images were captured on a Tecnai 12 transmission electron microscope operated at 80 kV with a Hamamatsu Orca HR CCD camera.

Cytoplasmic staining of TMEM106B by antibodies specific for its N-or C-termini
In order to determine whether TMEM106B aggregates contained the full-length protein, we used antibody A303-439A [9,22,36,49], which was raised against the N-terminal 50 amino acids of human TMEM106B, to stain tissue sections from the frontal cortex of a 25-and a 75-year-old neurologically normal individuals (Fig. 1, both cases were previously described [39]).Only diffuse cytoplasmic staining was observed.Similar results were obtained when we used antibody TMEM263, which was raised against a peptide corresponding to residues 263-274 of human TMEM106B which showed co-localization with endo-lysosomes (Fig. 1a, b; S6).No staining was observed following pre-adsorption of the antibody with TMEM106B (263-274) peptide (Fig. S1c).As previously reported [39], inclusions were identified using antibody TMEM239 (Fig. 1a, b; S6); they were also labelled by antibodies TMEM193, AP22247b and, as previously reported, antibody NB1-91311 [49] (Fig. S3).Staining of the frontal cortex from a 25-year-old individual lacking TMEM106B inclusions, similarly to the staining of the frontal cortex from the 75-yearold person with TMEM106B inclusions, showed diffuse cytoplasmic staining with A303-439A and TMEM263 (Fig. 1).A total of 28 brains from subjects aged 20-98 years old were stained with TMEM263, but no TMEM106B inclusions were detected by this antibody, and two representative cases are shown in Fig. 1.Antibodies A303-439A and TMEM263 also failed to recognise the 29 kDa band that was detected by antibody TMEM239 and is associated with the presence of TMEM106B aggregates (Fig. S2b).

TMEM106B inclusions are present in central and peripheral nervous systems, but not in peripheral organs
Using antibody TMEM239, we used tissues from a 78-year-old individual with PSP and a 90-year-old individual with AD to investigate the presence of TMEM106B inclusions in central nervous system regions other than the frontal cortex (Fig. 2).As shown in Fig. 2, optic nerve, temporal cortex, occipital cortex, hippocampus, midbrain, basal ganglia, pons/ medulla, cerebellum and spinal cord exhibited abundant TMEM106B inclusions.The same was also true of satellite cells in dorsal root ganglia, establishing the presence of TMEM106B inclusions in the peripheral nervous system (Fig. 2b).No inclusions were detected in heart, liver, spleen or hilar lymph nodes of neurologically normal individuals aged 71, 75 and 76 years with abundant TMEM106B inclusions in the brain (Fig. 3) or of a 49 year-old familial AD patient whose lung, gut, thyroid, and adrenal gland were also analysed (case 46, Supplementary Table 1).Young individuals were devoid of TMEM106B aggregates in all central and peripheral tissues examined (Supplementary Table 1).
So far, TMEM106B inclusions have been described in humans, we questioned whether they could also be present in aged mice.To this end we investigated their presence in the brains of 3-to 24-month-old wild-type and 7-month-old homozygous transgenic P301S tau mice with abundant tau pathology.No TMEM106B aggregates were detected (Fig. S5).

Immunogold electron microscopy of TMEM106B filaments in brain sections
Immunogold electron microscopy revealed the presence of numerous bundles of filaments in the neuropil of the neocortex (Fig. 7).These filaments were seen in cell body (Fig. 7a,b) and processes (Fig. 7c) of cells that had the characteristics of astrocytes (nucleus without an evident nucleolus and sparse chromatin; little endoplasmic reticulum in the cytoplasm).Decorated filaments could be found within structures resembling secondary endolysosomes and in vacuoles (Fig. 7a).The immunogold labelling was specific in that gold particles were not observed in association with other cellular structures.In agreement, double immunofluorescence showed that some TMEM106B inclusions localised to lysosomes (Fig. S6a).No filament decoration was observed with antibody TMEM263.

Discussion
Abundant filaments made of residues 120-254 of TMEM106B form in an age-dependent manner in human brains [33,39,43,49], including those from individuals with various neurodegenerative conditions, such as AD, PD, DLB, FTDP-17T, FTLD-TDP, limbic-predominant age-related TDP-43 encephalopathy (LATE), pathological ageing and age-related tau astrogliopathy [3,8,14,21,23,29,39,43,49].It remains to be seen if the formation of filaments of TMEM106B can influence the development and/or progression of disease.It remains to be determined if the formation of TMEM106B filaments can influence the risk of developing neurodegenerative diseases.Lysosomal dysfunction is commonly observed during ageing and in neurodegenerative diseases [10] and it has been suggested that this may be a consequence of TMEM106B aggregation [24], although it is also possible that lysosomal dysfunction leads to TMEM106B aggregation.Interestingly, loss of TMEM106B has been shown to exacerbate TDP-43 and tau pathologies, as well as neurodegeneration in transgenic mice [13,15,17,44,52].Many studies have focused on diseases with TDP-43 aggregates, for which the genetic association with TMEM106B was first shown [42].Cases with the TMEM106B risk allele develop more TMEM106B inclusions [23,28,29,33], although the influence on disease development remains uncertain.Unfortunately, the TMEM106B haplotype was not known in the cases investigated here, because they were obtained from brain banks as paraffin-embedded tissue sections with no DNA or fresh tissues available.However, we report that young people with either a lysosomal storage disorder, mitochondrial dysfunction or abundant protein aggregates, as in JOS, familial AD and Down syndrome, did not show TMEM106B aggregates.We cannot exclude that this could be the case of young individuals with GRN mutations for whom the TMEM106B haplotype is relevant for disease manifestation.This may become possible to investigate while the patient is still alive, given the recent finding that the PET ligand PBB3 recognises TMEM106B aggregates [50].
By immunohistochemistry, TMEM106B inclusions were most abundant in astrocytes, as reported previously [22,28,33,49,50], but they were also present in other cells, including neurons.In dorsal root ganglia, inclusions were most abundant in glial cells.This is interesting considering that previous studies reported higher TMEM106B protein expression in neurons and oligodendrocytes than astrocytes [16].The origin of TMEM106B aggregates in astrocytes remains to be determined.
TMEM106B is a transmembrane protein of 274 amino acids that is found in late endosomes/lysosomes  [16].Filament formation requires cleavage in the intraluminal domain between residues 119 and 120.Here we report that antibody TMEM263, which recognises the C-terminal part of recombinant TMEM106B on immunoblots (Fig. S2) and co-localises with lysosomes (Fig. S6), failed to recognise inclusions by immunohistochemistry, immunoblotting and immuno-EM, suggesting that residues 263-274 of TMEM106B are not part of the filaments.
TMEM263 gave diffuse cytoplasmic staining in brain cells, as did A303-439A, an antibody specific for the N-terminal region of TMEM106B.Similar diffuse staining for N-terminal and C-terminal antibodies has previously been reported [7,9,22,36,49].Both antibodies gave similar staining, irrespective of age, as we showed in brain cells from 25-and 75-year-old individuals [39].TMEM239 labelled inclusions in the brain from the 75-year-old individual [39], as well as in the brains from other aged subjects.However, TMEM106B aggregates were not found in aged mice.
TMEM106B filaments may lack a fuzzy coat, unlike filaments made of tau [18,40], α-synuclein [38,47], TDP-43 [2,3] and amyloid-beta [46,48].This is also supported by mass spectrometry results that failed to detect C-terminal peptides, including residues 255-262, in brain extracts [3,14].Lack of a fuzzy coat could influence the spreading and toxicity of assembled TMEM106B [41].Whether TMEM106B filaments can exhibit prion-like properties is not known.Not only may this require two cleavages, but the reported stability of TMEM106B filaments [23,35] may limit their spreading since the prion-like properties are inversely related to the stability of assembled proteins [11].
The formation of TMEM106B inclusions in the brain is age-dependent and disease-independent [39,43].As shown here, sensory ganglia and spinal cord also contained TMEM106B aggregates, while no inclusions were detected in heart, liver, spleen or hilar lymph nodes of subjects with brain aggregates.Moreover, TMEM106B inclusions were not present in the central nervous system of young individuals, even in the presence of disease.
By immunoelectron microscopy of cortical sections from a case of AD with abundant TMEM106B inclusions, we found filaments decorated by antibody TMEM239 that were closely associated with secondary lysosomes and vacuoles.This suggests that TMEM106B may assemble first in the organelles that normally express the native protein or that assembly can occur after escaping the damaged lysosomes.
In conclusion, we show that TMEM106B filaments are stained by amyloid dyes and confirm that they are predominantly present in non-neuronal cells.TMEM106B aggregates were not present in several peripheral human organs; similarly, they were also absent from wild-type and transgenic mouse brains.By immunogold-EM aggregates were found to be associated with lysosomes and vacuolar structures.By light microscopy, the inclusions were labelled by an antibody against the core region of TMEM106B filaments, but not by an antibody against the Fig. 7 Immunogold electron microscopy on brain tissue sections.Immunogold EM on frontal cortex of an individual aged 90 years with Alzheimer's disease (case 4) revealed the presence of bundles of filaments within the cell body and processes of cells, most likely astrocytes.The bundles of filaments decorated by gold particles were seen in association with dense osmiophilic structures with the morphological characteristics of secondary lysosomes.Bundles of filaments decorated by the gold particles, as well as the dense osmiophilic structures, may be present in vacuoles and were surrounded by a membrane.a Low power transversal view of a cell process containing immunogold labelled filaments enlarged in Box (a1).Adjacent to the transversal view of the cell process, another bundle of filaments decorated by gold particles is indicated by two arrows; a1, higher magnification of the smaller osmiophilic structure associated with the bundle of gold-decorated filament.b Low power view of a portion of a cell body.Part of the nucleus is seen in the upper right.Box (b1) shows an electron-dense osmiophilic structure with vacuoles that is associated with a bundle of filaments decorated by gold particles.Another bundle of filaments is indicated by two arrows; b1 higher magnification of the electron-dense osmiophilic structure associated with filaments decorated by gold particles.c Low power view of a cell process containing two bundles of filaments, one in box (c1) and the other indicated by two arrows; c1, higher magnification of filaments seen in box (c).Scale bar in c, 1 µm (also for a and b) and in c1, 200 nm (also for a1 and b1) C-terminus of TMEM106B.It remains to be determined what roles these inclusions may have in the brains of aged human individuals.

Fig. 1
Fig. 1 Immunohistochemical staining with anti-TMEM106B antibodies A303-439A, TMEM239 and TMEM263.a, Schematic of human TMEM106B, with the epitopes of anti-TMEM106B antibodies indicated.b, Staining of frontal cortex sections from neurologically normal individuals aged 75 (case 1) and 25 (case 2) years (yo = years-old).Nuclei are counterstained in red.Scale bars: 50 µm and 20 µm in the inset.Note similar cytoplasmic staining by A303-439A and TMEM263 in 25 yo and 75 yo individuals; TMEM239 stained inclusions in the 75 yo individual, with no staining in the 25 yo individual

Fig. 2
Fig. 2 Presence of TMEM106B inclusions in central and peripheral nervous systems.a, Staining with antibody TMEM239 of different brain regions from an individual aged 78 years with progressive supranuclear palsy (case 3).Scale bar, 50 µm.b, Staining with antibody TMEM239 of brain, spinal cord and dorsal root ganglion sections from an individual aged 90 years with sporadic Alzheimer's disease (case 4).Scale bar, 50 µm.Arrows in the dorsal root ganglion picture indicate TMEM106B aggregates

Fig. 3
Fig. 3 Absence of TMEM106B inclusions in peripheral organs.a Staining with TMEM239 of heart, liver, hilar lymph node, spleen and brain sections from neurologically normal individuals aged 71 (case 4), 75 (case 1) or 76 (case 6) years (yo = years-old).Nuclei are counterstained in red.Note the presence of TMEM106B inclusions in brain, but not in peripheral organs.Lipofuscin was observed as brown-coloured grainy material in some peripheral organs.Immunostaining of brain for the 75 yo subject is shown in Fig. 1. b Immunoblot analysis with TMEM239 of sarkosyl-insoluble extracts from heart, liver, hilar lymph node, spleen and brain from the same neurologically normal individuals aged 71, 75 or 76 years (yo = years-old) as in a.Note the presence of a 29-kDa band only in brain (red arrow).No brain tissue from the 71 yo individual was available for immunoblotting, but the presence of TMEM106B inclusions in brain is shown by immunostaining in a

Fig. 5
Fig. 5 TMEM106B inclusions in brain cells.a Double-immunostaining of TMEM239 (black) and neuronal marker NeuN (red) of cortical sections from a neurologically normal individual aged 84 years (case 23); a1, a2, a3, close-up images of insets in a. TMEM106B inclusions were sometimes present in NeuN-positive nerve cells.b Double-immunostaining of TMEM239 (black) and astrocytic marker glial fibrillary acidic protein (GFAP) (red) of cortical sections from a neurologically normal individual aged 76 years (case 24); b1, b2, b3, close-up images of insets in b.Many TMEM106B inclusions were present in GFAP-positive astrocytes.c Double-immunostaining of TMEM239 (black) and oligodendrocytic marker APC/CC1 (red) of cortical sections from a neurologically normal individual aged 76 years (case 24); c1, c2, c3, close-up images of insets in c.TMEM106B inclusions were occasionally present in APC/CC1-positive oligodendrocytes.d Double-immunostaining of TMEM239 (black) and microglial marker Iba-1 (red) of cortical sections from a neurologically normal individual aged 84 years (case 23); d1, d2, d3, close-up images of insets in d.TMEM106B inclusions were occasionally present in Iba-1-positive microglia

Fig. 6
Fig. 6 Double-labelling immunofluorescence of TMEM106B antibodies and luminescent conjugated oligothiophenes (LCOs), which are amyloid dyes.a Double-labelling immunofluorescence of sections from the frontal cortex of neurologically normal individuals aged 83 (case 25) and 84 (case 23) years with antibody TMEM239 (red) and LCOs pFTAA, hFTAA, HS68 and Amytracker 540 (green).Inclusions were labelled by both TMEM239 and LCOs (double labelling in yellow).b, c Double-labelling immunofluorescence of sections from the frontal cortex of a neurologically normal individual aged 84 years (case 26) using pFTAA and anti-TMEM106B antibodies TMEM263 (b) and A303-439A (c) (recognising TMEM106B C-terminus and N-terminus respectively).Inclusions were only labelled by pFTAA.d Double-labelling immunofluorescence of sections from the dorsal root ganglion of an individual aged 90 years with sporadic Alzheimer's disease (case 4) with antibody TMEM239 (red) and pFTAA, (green).Inclusions were labelled by both TMEM239 and pFTAA (double labelling in yellow)