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TMEM106B aggregation in neurodegenerative diseases: linking genetics to function
Molecular Neurodegeneration volume 18, Article number: 54 (2023)
Abstract
Background
Mutations of the gene TMEM106B are risk factors for diverse neurodegenerative diseases. Previous understanding of the underlying mechanism focused on the impairment of lysosome biogenesis caused by TMEM106B loss-of-function. However, mutations in TMEM106B increase its expression level, thus the molecular process linking these mutations to the apparent disruption in TMEM106B function remains mysterious.
Main body
Recent new studies reported that TMEM106B proteins form intracellular amyloid filaments which universally exist in various neurodegenerative diseases, sometimes being the dominant form of protein aggregation. In light of these new findings, in this review we systematically examined previous efforts in understanding the function of TMEM106B in physiological and pathological conditions. We propose that TMEM106B aggregations could recruit normal TMEM106B proteins and interfere with their function.
Conclusions
TMEM106B mutations could lead to lysosome dysfunction by promoting the aggregation of TMEM106B and reducing these aggregations may restore lysosomal function, providing a potential therapeutic target for various neurodegenerative diseases.
Background
Mutations of TMEM106B have been identified as genetic risk factors for neurodegenerative diseases including Frontotemporal lobar degeneration (FTLD) [1] and limbic-predominant age-related TAR DNA binding protein 43 (TDP-43) encephalopathy [2]. TMEM106B has also been reported to modulate patients’ cognitive functions in other neurodegenerative diseases, such as Alzheimer’s Disease (AD) [3], Parkinson’s Disease (PD) [4], and amyotrophic lateral sclerosis (ALS) [5]. The classic studies of TMEM106B function indicated that this protein is an important regulator for lysosomal function [6]. Thus, the disease-related TMEM106B genetic polymorphisms could contribute to pathogenesis by disrupting lysosome functions. This view has recently been challenged by a series of reports. These studies showed that TMEM106B aggregation is a widespread pathology that exists in the post-mortem brain tissues of diverse neurodegenerative diseases, including FTLD, PD, AD, ALS and multiple system atrophy (MSA) [7,8,9,10]. These findings suggest that TMEM106B can form protein aggregation that may be contributing to the neurodegeneration. The collection of these studies points out that the underlying mechanism linking TMEM106B mutation and disease onset could be more than the loss-of-function for regulating lysosome biogenesis, but could be a gain of toxicity due to the formation of protein aggregates. In light of this new possibility, a re-evaluation of previous opinion of TMEM106B’s function and association with neurodegenerative diseases is needed.
Main text
TMEM106B is associated with clinical characteristics of neurodegenerative diseases
Mutations in TMEM106B are risk factors for diverse neurodegenerative diseases
Human TMEM106B gene is located on chromosome 7p21, with nine exons. The most well studied single-nucleotide polymorphism (SNP) rs1990622 is in a non-coding area that could play a regulatory role. The T allele at this position is considered the major isoform (T/C frequency is 0.58/0.42 in Caucasian population and 0.37/0.63 in Asian) [11]. The major T allele has been linked to higher risks for developing neurodegenerative diseases or exacerbated cognitive decline, whereas the minor C allele is associated with a protective phenotype. In addition, one coding variant of TMEM106B, Thr185Ser encoded by SNP rs3173615 (C/G 0.60/0.40 in Caucasian and 0.37/0.63 in Asian), has been reported to be protective against several neurodegenerative disorders [12, 13]. We summarized these human association studies below.
The most robust associations between TMEM106B polymorphism and the development of diseases have been reported in diseases in which TDP-43 is the major proteinopathy in the brain. For example, TDP-43 inclusion bodies are the primary aggregation found in a major subtype of frontotemporal lobar degeneration (FTLD-TDP) patients. Also, genome-wide association studies found that the major T allele of SNP rs1990622 was linked to an increased FTLD-TDP risk (odds ratio: 1.64), whereas the minor C allele was protective (odds ratio 0.61) [14,15,16]. The allele rs1990621 has been reported to be associated with neuroprotective effects among FTLD patients [3]. Among people carrying Progranulin gene (GRN) mutations, which are known to cause FTLD, TMEM106B SNP rs1990622 could further increase the risk for FTLD-TDP, potentially by modulating GRN levels [14, 15, 17]. On the other hand, studies found no relationship between rs1990622 and subtypes of FTLD without the TDP-43 pathology [18], suggesting that the interaction with TDP-43 could be important for the pathogenesis effect of TMEM106B SNPs.
Consistent with this view, Limbic-predominant age-related TDP-43 encephalopathy (LATE) is another disease with prominent TDP-43 proteinopathy that shows robust association with TMEM106B polymorphism. LATE patients present an amnestic dementia syndrome that resembles AD [19], and autopsy studies have revealed that LATE patients show prevalent TDP-43 proteinopathy, some with concurrent hippocampal sclerosis. All subtypes of LATE with distinct clinical patterns are associated with TMEM106B rs1990622 polymorphism (OR = 3.3), suggesting that TMEM106B serves as an independent risk factor for LATE [2].
TMEM106B polymorphism showed no or weak association with the risk for ALS [5], AD [20] or PD [4]. Interestingly, the leading brain proteinopathy in AD and PD patients are not TDP-43 (namely, amyloid plaques and neurofibrillary tangles in AD and α-synuclein in PD) [21]. Regarding ALS, although TDP-43 is the major protein deposition, the primary symptom of motor dysfunction is likely due to the damage of motor neurons in the spinal cord [22, 23]. These data strongly suggest that the pathological effect of TMEM106B’s polymorphism can be modulated by the interaction with TDP-43 in the brain. While the molecular mechanism underlying this interaction is currently unknown, one possibility is that TMEM106B facilitates the aggregation of TDP-43 in the brain [24], which causes downstream cytotoxicity [25,26,27].
If the scenario described above is true, TMEM106B could still contribute to the brain function decline in AD, PD and ALS, even though the primary clinical symptoms of these diseases are not related to the TDP-43 depositions in the brain. Consistent with this view, several SNPs of TMEM106B have been reported to correlate with cognitive decline in AD, PD and ALS. Specifically, TMEM106B protective alleles rs1990622C are associated with slower deterioration of language function in ALS patients [5]. In AD, analysis showed TMEM106B regulates genetic pathways that converge with those affected by APOE-amyloid-β interaction [28]. TMEM106B rs1990621 variation has also been reported to correlate with neuronal proportion [3]. TMEM106B rs1990621 variation has also been reported to correlate with levels of neurofilament light chain in the cerebrospinal fluid of AD patients [29], which is a strong indicator of neurodegeneration. Among PD patients, rs1990622T carriers exhibit faster longitudinal decline in cognition, indicating that TMEM106B functions as a genetic modulator for cognitive trajectory in PD [4].
Together, these studies of genetics showed that TMEM106B variants are associated with the onset or clinical manifestation of major neurodegenerative diseases, highlighting the importance of the interaction with TDP-43 in the brain. Next, we turn to examine the structural features of TMEM106B aggregates found in recent pathology studies.
TMEM106B protein depositions exist in a wide range of neurodegenerative diseases
Jiang et al. serendipitously discovered that all amyloid fibrils isolated from post-mortem brain tissues of four FTLD-TDP patients showed negative labeling of anti-TDP-43 antibody [10]. Subsequent immunogold labelling and cryo-electron microscopy revealed that these amyloid fibrils were made up with TMEM106B [10]. This finding was unexpected as TMEM106B was not previously reported to form amyloid fibrils. Several other studies also identified TMEM106B fibrils in diverse neurodegenerative diseases such as FTLD-TDP, tauopathy, AD, and α-synucleinopathy [7,8,9], and normal aging [9], indicating that TMEM106B fibrils are a previously unappreciated common protein aggregation that exists in the brain (Fig. 1). Interestingly, when compared to age-matched controls, the burden of TMEM106B fibrillization is much higher in most individuals with neurodegeneration [7, 9, 30], suggesting that the TMEM106B fibrils may not be a benign structure, but could exert a toxic effect and contribute to age-dependent neurodegeneration, although no direct evidence currently exists.
TMEM106B depositions have been identified in different brain regions. Frontal lobe area is the most common region to date to show TMEM106B deposits. Studies have reported TMEM106B filament in frontal lobe in patients with familial AD, early onset AD, PD, FTLD-TDP, corticobasal degeneration, limbic-predominant neuronal inclusion body 4R tauopathy, dementia with Lewy bodies, progressive superanuclear palsy and normal aging [7,8,9,10]. In addition, deposits have been reported in the motor cortex of ALS patients [9]. TMEM106B fibrils can also form in subcortical regions, including nucleus accumbens, hippocampus, cingulate gyrus, amygdala, putamen and caudate [7]. Overall, many aspects of the TMEM106B pathology remain unknown, probably due to limited post-mortem brain tissue from patients. At this point, while several studies reported that TMEM106B fibrils do exist in many brain regions, whether there is a region- or diseases- specific susceptibility for TMEME106B aggregation and how these aggregations evolve or spread longitudinally with the development of the diseases, require further investigation.
Cryo-EM studies have resolved that the TMEM106B fibrils are aggregates from the C-terminus fragments (S120-G254) [7,8,9,10]. Furthermore, researchers identified three major isoforms of TMEM106B fibrils. The three isoforms differ in their structure of the middle region (A167 – M210), in which type I forms a loose amphipathic cavity, and type II and type III form increasingly tighter structures. In addition, the same fibril isoform can connect to each other via the sidechains of residues K178 and R180 to form doublet fibril [7]. In most cases, the ratio between singlet and doublet fibrils varies between 0.5 and 2. Importantly, the type II and type III fibrils are particularly enriched in patients with neurodegenerative diseases, while type I and II can be found in brains with normal aging [8]. These structural data again indicated that a distinct isoform of TMEM106B may be associated with neurotoxicity [8].
Physiological function of TMEM106B in regulating lysosome functions
Introduction of TMEM106B
TMEM106B is a single-pass, type 2 integral membrane glycoprotein with 274 residues that predominantly locates in the membranes of late endosomes and lysosomes [31]. TMEM106B shows robust colocalization with the late endosome and lysosome markers Rab7, cathepsin D, and LAMP1, and relatively poor colocalization with the early endosome marker Rab5 and the recycling endosome marker Rab11, indicating that late endosomes and lysosomes are the primary subcellular location of TMEM106B [6].
TMEM106B protein sequence contains three structural domains. The N-terminal region consists of residues 1–96, which extends into the cytoplasm, can interact with both itself and TMEM106C, forming homopolymer or hetero-multimers at the lysosome surface [32]. A single-pass helix transmembrane domain can be found at residues 97–117. A C-terminal region of residues 118–274 exists in the lumen of lysosomes. This domain contains five important N-glycosylation sites. For TMEM106B to be transported outside of the endoplasmic reticulum and into late-stage cellular compartments, glycosylation is necessary. Whether endogenous and transgenic overexpressed TMEM106B, they all localize to late endosomes and lysosomes [31]. Post-translational modifications have been reported on TMEM106B to modulate its function. Glycosylation of residues N183 and N256 have been reported to regulate lysosome localization and TMEM106B degradation [31]. In addition, the proteolytic processing of TMEM106B also occurs in the C-terminal region, which produces the fragment that forms the protein aggregation [30, 33].
TMEM106B regulates morphology, acidification and transport of lysosome
Previous studies reported that TMEM106B could regulate various aspects of lysosome functions. The N-terminal region of the protein can interact with the clathrin heavy chain (CTLC), the μ1 subunit of adipocyte protein 2 (AP2M1), and endocytic adaptor proteins, indicating that TMEM106B may be crucial for the endolysosome sorting process [32]. In addition, TMEM106B N-terminus can also interact with microtubule-associated protein 6 (MAP6), suggesting an important function in controlling the retrograde transport of lysosomes [34]. The C-terminal region of TMEM106B can interact with lysosomal protease cathepsin D [35] and proton pump V-ATPase [36], thus pointing to a modulatory role of TMEM106B for lysosome acidification and protein degradation.
Consistent with these biochemistry characterizations, TMEM106B knockout led to a severe disruption of lysosome functions. TMEM106B knockdown reduces the total number of lysosomes in the cells [32]. The remaining lysosomes change from the normal cytoplasmic localization to an abnormal clustering at the axon initial segment or perinuclear space [32, 37]. At the same time, the morphology of lysosomes dramatically enlarges into a vacuole-like shape when lacking TMEM106B [38]. This is accompanied by a disruption of lysosome maturation, as TMEM106B knockdown resulted in less efficient fusion with autophagosome, poor protein degradation efficiency, and insufficient acidification [36, 39]. Thus, TMEM106B plays important roles in the transportation and maturation of lysosomes and is necessary for the physiological biogenesis of this organelle (Fig. 2).
Physiological processes regulated by TMEM106B
The Tmem106b knockout models show several pathological phenotypes, which may be ultimately attributed to altered lysosomal functions. In cultured neurons, lacking TMEM106B causes an imbalance between retrograde and anterograde transportation of lysosomes, leading to an abnormal accumulation of lysosomes around the soma. Blocking the overly active retrograde transportation can partially reverse the reduction of dendritic branching in cultured cells, indicating a restoration of lysosomal function. The general reduction of lysosome function in TMEM106B knockdown is also indicated by a build-up of lipofuscin [37], and an alteration of TFEB-related genetic pathways [40]. Importantly in mice, the hippocampus and cerebellum are the brain regions with high levels of Tmem106b expression in the brain [41], thus these two regions might be the most susceptible to the loss of TMEM106B function. Indeed, in Tmem106b knockout mice, the most severe disruption of lysosome function and cytotoxicity happens in the Purkinje cells, while cortical neurons show only mild changes [42]. In humans, the expression of TMEM106B is more universal cross different brain regions [43], suggesting that lacking TMEM106B may affect more broadly.
In addition to neurons, TMEM106B is also expressed in oligodendrocytes. TMEM106B deficiency results in myelination abnormalities including the separation and vacuolization of myelin sheaths [35]. At the same time, the numbers of matured oligodendrocytes are reduced [44]. The abnormal myelination in Tmem106b knockout mice may also be due to the change in lysosome functions. This is because the integration of major protein components, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), to myelin sheath requires lysosome exocytosis [45, 46]. Lacking functional TMEM106B disrupts the normal trafficking of PLP to cell surface and induces perinuclear clustering of lysosomes [35], indicating a disruption of oligodendrocyte maturation [44]. Tmem106b knockout also leads to a general reduction in lysosomal proteins including cathepsin D in oligodendrocytes [35], which is known to be important for PLP processing [47].
While other glia cells also express Tmem106b, including astrocytes, microglia and endothelia cells, the function of Tmem106b in these cells are less clear. In Tmem106b knockout mice, no obvious lysosomal phenotypes were observed in microglia and astrocyte in the young Tmem106b−/− mice [42], suggesting a different protein may exist in these cells to regulate lysosome biogenesis. Also, there is an upregulation of inflammatory genes in Tmem106b knockout mice, indicating that astrocytes and microglia could be activated [39]. In another study, TMEM106B deficiency in mice lead to reduced microglia proliferation and activation and increased microglial apoptosis in response to demyelination [48]. It is possible that the inflammatory phenotype in these cells is due to a secondary response to the ongoing cytotoxicity in neurons and oligodendrocytes.
Disease-associated variants increase TMEM106B levels
TMEM106B variant increase TMEM106B level
The most well-studied TMEM106B variant rs1990622 is located in the non-coding region of the gene, which does not change the sequence or structure of the protein product. A recent study revealed that this region binds to a transcription factor CTCF (CCCTC-binding factor) and regulates the activity of TMEM106B promoter [49]. The disease-promoting variant rs1990620A increases the expression of TMEM106B [49]. Thus, it is possible that increasing levels of TMEM106B might be contributing to the higher risks for developing neurodegenerative diseases [1, 43]. This view is also supported from the studies examining the effect of another important SNP rs3173615, which affects the 185th amino acid of the protein. This position is close to sites of glycosylation in the C-terminal region of the protein, which are important for the localization of TMEM106B to lysosomes [31]. Studies revealed that expressing the risk isoform T185 leads to a higher level of TMEM106B compared to that when expressing the protective isoform S185 [50]. Interestingly, suppressing lysosomal digestion blocks this effect, while stopping novel protein synthesis does not, indicating that the risk isoform T185 reduces the degradation of the protein, leading to a higher level of TMEM106B in cells [50]. Together, these studies link the elevated levels of TMEM106B with higher risk for developing neurodegenerative diseases.
Increasing TMEM106B levels disrupt lysosome function
The findings that higher levels of TMEM106B are associated with higher risk for developing neurodegenerative diseases suggests that the overexpression of TMEM106B might be a plausible model to study its involvement in the pathogenesis process. Interestingly, TMEM106B overexpression disrupts several aspects of lysosome function, similar to the phenotypes found with TMEM106B deficiency. In cell cultures, over expression of TMEM106B leads to the formation of abnormally large vacuoles and associated reduction in function [35, 51, 52], which are associated with reduced dynamism [32], less effective protein degradation [51] and poor acidification of lysosomes [35, 51]. Furthermore, humans carrying the rs1990622 risk allele (T/T) showed increased Purkinje neuron loss, mimicking the phenotype seen in TMEM106B knockout mice [42]. Bcl-xL, which is previously used for preventing caspase-dependent mitochondrial-mediated apoptosis, was proved to significantly ameliorate the neurotoxicity of TMEM106B overexpression [53]. Together, these data indicate a similar effect of lysosome dysfunction between TMEM106b deficiency and overexpression.
Increased TMEM106B levels could potentially promote its aggregation
Why would TMEM106B deficiency and overexpression both disrupt lysosome function? A previous hypothesis suggests that TMEM106B level is critical for normal lysosome function and an imbalance towards either direction could disrupt this delicate equilibrium. In a Tmem106b transgenic mouse model, total protein levels of TMEM106B remained unchanged despite increased amount of mRNA and protein expression [54]. While this provides evidence supporting tight control of TMEM106B in vivo, the molecular mechanism allowing deficiency and overexpression to show the same phenotype is still lacking.
In light of the recent findings that TMEM106B can form protein aggregates commonly seen in patients with neurodegenerative diseases, we here propose a model that reconciles the phenotype of TMEM106B deficiency and overexpression. The proteolytic processing of TMEM106B produces a C-terminal fragment in the lumen of lysosomes. In normal conditions, this fragment undergoes a lysosome-dependent degradation. However, when lysosome function is insufficient, or the levels of TMEM106B increased, the C-terminal fragments of TMEM106B will not be degraded in time, and can start the process of fibrilization. The TMEM106B fibrils may recruit or interact with normal TMEM106B proteins, interfering their functions in modulating lysosome biogenesis, thus effectively causing a deficiency of functional TMEM106B (Fig. 3).
Consistent with this hypothesis, previous studies have shown that lysosome activity can regulate the levels of TMEM106B. For example, acute treatment of Bafilomycin reduced lysosomal activities by interfering with lysosome acidification [55, 56]. Applying Bafilomycin significantly increased TMEM106B on protein levels through a predominant post-transcriptional mechanism [31]. Increased levels of TMEM106B are also reported in models of lysosomal storage disorder [57].
While our model remains speculative at present, several testable hypotheses can be made. A key process that links TMEM106B overexpression with a loss-of-function phenotype is a fibrilization process. We hypothesize that electron microscopy examination of tissue or cells with elevated expression of TMEM106B could reveal the existence of TMEM106B fibrils. In addition, we propose that TMEM106B fibrils could have ‘prion-like’ properties that recruits monomers. This is testable in vitro by seeding the TMEM106B monomer solution with extracted fibrils and examining the fibrilization over time. Furthermore, we propose that introducing TMEM106B fibrils will disrupt lysosome functions, which is readily testable in cultured cells. These experiments could provide mechanistic insights linking the overexpression phenotype due to disease-associated TMEM106B mutations and the TMEM106B fibrils universally found in patients’ brains.
Other risk factors interact with TMEM106B pathology
The association with aging
TMEM106B gene expression is associated with normal aging [58]. Recent studies demonstrate that TMEM106B forms amyloid fibrils in human brains in an age-dependent manner [7,8,9,10]. In addition, in patients with neurodegenerative diseases, the amount of TMEM106B fibrils are found to be higher than age-matched control brains [7]. These data indicate that the formation of TMEM106B fibrils exacerbated with aging, and can be additionally modulated by disease conditions. However, it is not clear whether the increased amount of TMEM106B fibrils in diseased brains is an indication of reduced lysosomal functions, or a cause for lysosomal dysfunction. It has been reported that aging is associated with reduced lysosomal acidification and protease activity [59, 60], which could contribute to the insufficient degradation of TMEM106B C-terminal fragment. On the other hand, the formation of TMEM106B fibrils, according to our model (Fig. 3), may be in turn depleting the endogenous TMEM106B and exacerbates defects in lysosome biogenesis. This vicious cycle could contribute to the accumulation of other protein aggregates in the cell, eventually leading to complete failure of the lysosome function and cell death.
The association with Progranulin (GRN mutation)
One of the major neurodegenerative diseases associated with TMEM106B is frontotemporal dementia (FTD). The loss-of-function mutation of the GRN is well-characterized to cause familial FTD [61]. Importantly, the risk for FTD in individuals with GRN mutations is further modulated by SNPs in the TMEM106B gene [15, 17], suggesting an additive effect of the mutations in these two genes. Since localization to lysosomes is important for progranulin function [62] and loss of progranulin could lead to lysosome storage disease [63, 64], it is possible that TMEM106B and GRN mutations both contribute to the development of FTD via worsening lysosome functions. Interestingly, a study showed that Tmem106B deletion and Grn deletion cause opposite changes in lysosome proteins, thus knocking out Tmem106b to a relatively low level could partially rescue the phenotype of GRN knockout mice [36]. However, this result remains controversial as several studies showed that deletion of Tmem106b on a background of GRN knockout further disrupts lysosome/autophage function as well as the health of the animal [39, 65, 66]. Furthermore, another study reported no benefitial effect of partial Tmem106b reduction on the social deficits and most lysosome abnormalities in Grn+/− mice [67]. Also knocking in the classic protective Tmem106bT186S variant (SNP rs3173615) did not exert protective effects in GRN knockout mice [68]. The inconsistent results regarding whether modulating TMEM106B levels is a viable therapeutic strategy for GRN-FTLD call for more studies.
Future direction based on TMEM106B pathology and therapy
TMEM106B is involved in development of neurodegenerative disease
Prior genetics studies revealed a robust relationship between several SNPs in the TMEM106B gene and the risk for developing neurodegenerative diseases. However, the biological mechanism underlying this association remains mysterious, since studies showed that TMEM106B is necessary for lysosome biogenesis, yet the disease-associated genotypes all lead to increased levels of TMEM106B. Given the recent reports that TMEM106B can form fibrils in the brain [7,8,9,10], we proposed a new model in this review as a plausible pathogenic mechanism: increased levels of TMEM106B may promote the formation of TMEM106B fibrils, which exerts a dominant negative effect on the endogenous TMEM106B and disrupts lysosome function. As described in the previous section, several specific predictions derived from this model can be tested and may elucidate the pathogenic mechanism of TMEM106B mutations.
Furthermore, under the framework of our model, lysosome dysfunction due to TMEM106B mutations could add to the formation of other protein aggregation, such as amyloid-β, α-synuclein, phosphorylated tau and TDP-43. Interestingly, genetics studies showed that TMEM106B mutations is particularly detrimental for diseases with TDP-43 being the primary type of protein aggregates in the brain (see Sect. "Mutations in TMEM106B are risk factors for diverse neurodegenerative diseases"), suggesting that the effect of TMEM106B mutation may have certain specificity that preferentially affects TDP-43 over other types of protein aggregation. Consistent with this, TMEM106B genotype has been proven to modify TDP-43 pathology independent of C9orf72 status in human cohorts and cellular model [24]. The mechanism underlying such specificity requires future investigations (Fig. 4).
Therapeutic opportunities
To date there are no studies that have targeted TMEM106B for therapeutic intervention. This is understandable as the pathogenic mechanism of TMEM106B is not completely known. It is questionable whether increasing or decreasing the levels of TMEM106B could offer therapeutic effects. Our hypothesized model could provide some new insights in this regard. We proposed that the formation of TMEM106B fibrils might be the key turning point that starts the downstream pathogenic cascade. Therefore, targeting this initial fibrilization process could be a viable therapeutic target. Specifically, we suggest the following strategies (Fig. 5).
Improve glycosylation
For TMEM106B locating accurately to late-stage cellular compartments, glycosylation is necessary. Post-translational modifications could mediate the structures of polymorphic fibrils by influencing their inter-protofilament interfaces [69], although currently direct biochemical measurements of fibrilization kinetics affected by glycosylation is lacking for TMEM106B. On the other hand, glycosylation guarantees the protein stability and degradation rate [70], which could change the availability of physiologically functional TMEM106B. Therefore, improving post translational glycosylation is a potential therapeutic approach.
Blocking the production of C-terminal fragment
The C-terminal fragment of TMEM106B is the monomer to form fibrils. Thus, suppressing the production of this fragment could be an effective way of blocking the fibrilization process. To preserve the homeostasis of membrane proteins, single pass transmembrane proteins undergo sequential processing that includes ectodomain shedding and intramembrane proteolysis. This fragment of TMEM106B is produced by lysosome proteases in the luminal domain followed by an intracellular cytosolic domain is produced by the signal peptide peptidase-like 2A (SPPL2a) family protease in the transmembrane area [71]. SPPL2a antagonist could be a target to specifically block the production of C-terminal fragment. Despite the fact that proteasomes typically work better for non-aggregated proteins, inclusions can still be eliminated if abnormal protein fragment creation is halted [72].
Interfering with the fibrilization process
It is possible that certain small molecules could block the β-sheet formation of the TMEM106B fibrils. This approach could prevent the disruption of the endogenous TMEM106B’s function, thus blocking the disruption of lysosome biogenesis.
Promoting the degradation of TMEM106B fibrils
Conformation-specific antibodies for the TMEM106B fibrils can be raised to facilitate the degradation of TMEM106B fibrils. However, since the antibody-mediated degradation likely depends on microglia in the brain, this approach maybe more effective for the clearance of extracellular TMEM106B fibrils. Therefore, this strategy might be preventing the spreading of the TMEM106B aggregation between cells, but have little effects in neurons already containing these fibrils.
Restoring lysosome function
Given that normal TMEM106B maintain the physiological processes of lysosome, TMEM106B pathology is often associated with a breakdown of lysosome stability. A recent study found that lysosome function could be repaired, and this process is heavily reliant on lysosomal membrane integrity via complex regulation [73]. Repairing the proper pH circumstance and proteolysis function serves as a promising strategy for halting the progress of disease.
Conclusions
In this review, we propose a novel mechanism for neurodegenerative diseases inspired by the widely identification of TMEM106B deposition. Genetic mutations trigger the aggregation of TMEM106B filaments and imbalance of lysosome physiology, aggravated by advancing age, genetic predisposition, and environmental factors. Lysosome dysfunction further aggravates TMEM106B accumulations. The collapse of cellular autophagy mechanisms result in not only hallmark protein aggregation but also blocked signaling and even apoptosis independently. This cycle is widely distributed in neurons and glial cells among all brain regions. Targeting these amyloid fibrils could be a promising strategy for restoring neuron or glia functions, delaying the progress of neurodegeneration. Since the structures of TMEM106B filament vary between subtypes of diseases, the conformational variations could be an indicator for disease progress. This model for choosing which patients will benefit most from early therapies targeting the lysosome in a precision medicine approach needs more precise evidence before it can be established.
Availability of data and materials
Not applicable.
Abbreviations
- AD:
-
Alzheimer’s disease
- FAD:
-
Familial Alzheimer’s disease
- FTLD:
-
Frontotemporal lobar degeneration
- ALS:
-
Amyotrophic lateral sclerosis
- MSA:
-
Multiple system atrophy
- SNP:
-
Single-nucleotide polymorphism
- TDP-43:
-
TAR DNA-binding protein 43
- CTLC:
-
Clathrin heavy chain
- AP2M1:
-
The μ1 subunit of adipocyte protein 2
- MAP6:
-
Microtubule-associated protein 6
- PLP:
-
Proteolipid protein
- MOG:
-
Myelin oligodendrocyte glycoprotein
- LATE:
-
Limbic-predominant age-related TDP-43 encephalopathy
- EOAD:
-
Sporadic early-onset Alzheimer’s disease
- PA:
-
Pathological aging
- CBD:
-
Corticobasal degeneration
- LNT:
-
Limbic-predominant neuronal inclusion body 4R tauopathy
- DLB:
-
Dementia with Lewy bodies
- FTD:
-
Frontotemporal dementia
- PSP:
-
Progressive superanuclear palsy
- FTDP-17 T:
-
Familial frontotemporal dementia and parkinsonism linked to chromosome 17 caused by MAPT mutations
- MSA:
-
Multiple system atrophy
- PD:
-
Parkinson’s disease
- ARTAG:
-
Aging-related tau astrogliopathy
- PDD:
-
Parkinson’s disease Dementia
- AGD:
-
Argyrophilic grain disease
- FPD:
-
Familial Parkinson’s disease
- SPPL2a:
-
Signal peptide peptidase-like 2A
References
Van Deerlin VM, Sleiman PMA, Martinez-Lage M, Chen-Plotkin A, Wang LS, Graff-Radford NR, et al. Common variants at 7p21 are associated with frontotemporal lobar degeneration with TDP-43 inclusions. Nat Genet. 2010;42(3):234–9.
Cykowski MD, Arumanayagam AS, Powell SZ, Rivera AL, Abner EL, Roman GC, et al. Patterns of amygdala region pathology in LATE-NC: subtypes that differ with regard to TDP-43 histopathology, genetic risk factors, and comorbid pathologies. Acta Neuropathol. 2022;143(5):531–45.
Li Z, Farias FHG, Dube U, Del-Aguila JL, Mihindukulasuriya KA, Fernandez MV, et al. The TMEM106B FTLD-protective variant, rs1990621, is also associated with increased neuronal proportion. Acta Neuropathol. 2020;139(1):45–61.
Tropea TF, Mak J, Guo MH, Xie SX, Suh E, Rick J, et al. TMEM106B Effect on cognition in Parkinson disease and frontotemporal dementia. Ann Neurol. 2019;85(6):801–11.
Vass R, Ashbridge E, Geser F, Hu WT, Grossman M, Clay-Falcone D, et al. Risk genotypes at TMEM106B are associated with cognitive impairment in amyotrophic lateral sclerosis. Acta Neuropathol. 2011;121(3):373–80.
Brady OA, Zheng Y, Murphy K, Huang M, Hu F. The frontotemporal lobar degeneration risk factor, TMEM106B, regulates lysosomal morphology and function. Hum Mol Genet. 2013;22(4):685–95.
Chang A, Xiang X, Wang J, Lee C, Arakhamia T, Simjanoska M, et al. Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases. Cell. 2022;185(8):1346-1355.e15.
Fan Y, Zhao Q, Xia W, Tao Y, Yu W, Chen M, et al. Generic amyloid fibrillation of TMEM106B in patient with Parkinson’s disease dementia and normal elders. Cell Res. 2022;32(6):585–8.
Schweighauser M, Arseni D, Bacioglu M, Huang M, Lövestam S, Shi Y, et al. Age-dependent formation of TMEM106B amyloid filaments in human brains. Nature. 2022;605(7909):310–4.
Jiang YX, Cao Q, Sawaya MR, Abskharon R, Ge P, DeTure M, et al. Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43. Nature. 2022;605(7909):304–9.
L. Phan, Y. Jin, H. Zhang, W. Qiang, E. Shekhtman, D. Shao, D. Revoe, R. Villamarin, E. Ivanchenko, M. Kimura, Z. Y. Wang, L. Hao, N. Sharopova, M. Bihan, A. Sturcke, M. Lee, N. Popova, W. Wu, C. Bastiani, M. Ward, J. B. Holmes, V. Lyoshin, K. Kaur, E. Moyer, M. Feolo, and B. L. Kattman. “ALFA: Allele Frequency Aggregator.” National Center for Biotechnology Information, U.S. National Library of Medicine. 2020, www.ncbi.nlm.nih.gov/snp/docs/gsr/alfa/.
Cherry JD, Mez J, Crary JF, Tripodis Y, Alvarez VE, Mahar I, et al. Variation in TMEM106B in chronic traumatic encephalopathy. Acta Neuropathol Commun. 2018;6(1):115.
van Blitterswijk M, Mullen B, Nicholson AM, Bieniek KF, Heckman MG, Baker MC, et al. TMEM106B protects C9ORF72 expansion carriers against frontotemporal dementia. Acta Neuropathol. 2014;127(3):397–406.
Pottier C, Zhou X, Perkerson RB, Baker M, Jenkins GD, Serie DJ, et al. Potential genetic modifiers of disease risk and age at onset in patients with frontotemporal lobar degeneration and GRN mutations: a genome-wide association study. The Lancet Neurology. 2018;17(6):548–58.
Finch N, Carrasquillo MM, Baker M, Rutherford NJ, Coppola G, Dejesus-Hernandez M, et al. TMEM106B regulates progranulin levels and the penetrance of FTLD in GRN mutation carriers. Neurology. 2011;76(5):467–74.
van der Zee J, Van Langenhove T, Kleinberger G, Sleegers K, Engelborghs S, Vandenberghe R, et al. TMEM106B is associated with frontotemporal lobar degeneration in a clinically diagnosed patient cohort. Brain. 2011;134(Pt 3):808–15.
Cruchaga C, Graff C, Chiang HH, Wang J, Hinrichs AL, Spiegel N, et al. Association of TMEM106B gene polymorphism with age at onset in granulin mutation carriers and plasma granulin protein levels. Arch Neurol. 2011;68(5):581–6.
Llibre-Guerra JJ, Lee SE, Suemoto CK, Ehrenberg AJ, Kovacs GG, Karydas A, et al. A novel temporal-predominant neuro-astroglial tauopathy associated with TMEM106B gene polymorphism in FTLD/ALS-TDP. Brain Pathol. 2021;31(2):267–82.
Nelson PT, Dickson DW, Trojanowski JQ, Jack CR, Boyle PA, Arfanakis K, et al. Limbic-predominant age-related TDP-43 encephalopathy (LATE): consensus working group report. Brain. 2019;142(6):1503–27.
Hu Y, Sun JY, Zhang Y, Zhang H, Gao S, Wang T, et al. rs1990622 variant associates with Alzheimer’s disease and regulates TMEM106B expression in human brain tissues. BMC Med. 2021;19(1):11.
Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med. 2004;10(S7):S10–7.
Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;314(5796):130–3.
Blokhuis AM, Groen EJN, Koppers M, van den Berg LH, Pasterkamp RJ. Protein aggregation in amyotrophic lateral sclerosis. Acta Neuropathol. 2013;125(6):777–94.
Mao F, Robinson JL, Unger T, Posavi M, Amado DA, Elman L, et al. TMEM106B modifies TDP-43 pathology in human ALS brain and cell-based models of TDP-43 proteinopathy. Acta Neuropathol. 2021;142(4):629–42.
Xu YF, Zhang YJ, Lin WL, Cao X, Stetler C, Dickson DW, et al. Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice. Mol Neurodegener. 2011;6:73.
Barmada SJ, Skibinski G, Korb E, Rao EJ, Wu JY, Finkbeiner S. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J Neurosci. 2010;30(2):639–49.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci U S A. 2009;106(18):7607–12.
Yang HS, White CC, Klein HU, Yu L, Gaiteri C, Ma Y, et al. Genetics of Gene Expression in the Aging Human Brain Reveal TDP-43 Proteinopathy Pathophysiology. Neuron. 2020;107(3):496-508.e6.
Hong S, Dobricic V, Ohlei O, Bos I, Vos SJB, Prokopenko D, et al. TMEM106B and CPOX are genetic determinants of cerebrospinal fluid Alzheimer’s disease biomarker levels. Alzheimer’s & Dementia. 2021;17(10):1628–40.
T Vicente C, Perneel J, Wynants S, Heeman B, Van den Broeck M, Baker M, et al. C-terminal TMEM106B fragments in human brain correlate with disease-associated TMEM106B haplotypes. Brain. 2023;awad133.
Lang CM, Fellerer K, Schwenk BM, Kuhn PH, Kremmer E, Edbauer D, et al. Membrane orientation and subcellular localization of transmembrane protein 106B (TMEM106B), a major risk factor for frontotemporal lobar degeneration. J Biol Chem. 2012;287(23):19355–65.
Stagi M, Klein ZA, Gould TJ, Bewersdorf J, Strittmatter SM. Lysosome size, motility and stress response regulated by fronto-temporal dementia modifier TMEM106B. Mol Cell Neurosci. 2014;61:226–40.
Perneel J, Neumann M, Heeman B, Cheung S, Van den Broeck M, Wynants S, et al. Accumulation of TMEM106B C-terminal fragments in neurodegenerative disease and aging. Acta Neuropathologica. 2023;145(3):285–320.
Schwenk BM, Lang CM, Hogl S, Tahirovic S, Orozco D, Rentzsch K, et al. The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes. EMBO J. 2014;33(5):450–67.
Feng T, Sheng RR, Solé-Domènech S, Ullah M, Zhou X, Mendoza CS, et al. A role of the frontotemporal lobar degeneration risk factor TMEM106B in myelination. Brain. 2020;143(7):2255–71.
Klein ZA, Takahashi H, Ma M, Stagi M, Zhou M, Lam TT, et al. Loss of TMEM106B Ameliorates Lysosomal and Frontotemporal Dementia-Related Phenotypes in Progranulin-Deficient Mice. Neuron. 2017;95(2):281-296.e6.
Lüningschrör P, Werner G, Stroobants S, Kakuta S, Dombert B, Sinske D, et al. The FTLD Risk Factor TMEM106B Regulates the Transport of Lysosomes at the Axon Initial Segment of Motoneurons. Cell Rep. 2020;30(10):3506-3519.e6.
Stroobants S, D’Hooge R, Damme M. Aged Tmem106b knockout mice display gait deficits in coincidence with Purkinje cell loss and only limited signs of non-motor dysfunction. Brain Pathol. 2021;31(2):223–38.
Feng T, Mai S, Roscoe JM, Sheng RR, Ullah M, Zhang J, et al. Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice. EMBO Rep. 2020;21(10): e50219.
Kundu ST, Grzeskowiak CL, Fradette JJ, Gibson LA, Rodriguez LB, Creighton CJ, et al. TMEM106B drives lung cancer metastasis by inducing TFEB-dependent lysosome synthesis and secretion of cathepsins. Nat Commun. 2018;9(1):2731.
Allen Institute for Brain Science. Allen Mouse Brain Atlas . Available from mouse.brain-map.org. Allen Institute for Brain Science (2011). 2004.
Feng T, Luan L, Katz II, Ullah M, Van Deerlin VM, Trojanowski JQ, et al. TMEM106B deficiency impairs cerebellar myelination and synaptic integrity with Purkinje cell loss. Acta Neuropathol Commun. 2022;10(1):33.
Busch JI, Martinez-Lage M, Ashbridge E, Grossman M, Van Deerlin VM, Hu F, et al. Expression of TMEM106B, the frontotemporal lobar degeneration-associated protein, in normal and diseased human brain. Acta Neuropathol Commun. 2013;1:36.
Zhou X, Nicholson AM, Ren Y, Brooks M, Jiang P, Zuberi A, et al. Loss of TMEM106B leads to myelination deficits: implications for frontotemporal dementia treatment strategies. Brain. 2020;143(6):1905–19.
Winterstein C, Trotter J, Krämer-Albers EM. Distinct endocytic recycling of myelin proteins promotes oligodendroglial membrane remodeling. J Cell Sci. 2008;121(Pt 6):834–42.
Trajkovic K, Dhaunchak AS, Goncalves JT, Wenzel D, Schneider A, Bunt G, et al. Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites. J Cell Biol. 2006;172(6):937–48.
Guo DZ, Xiao L, Liu YJ, Shen C, Lou HF, Lv Y, et al. Cathepsin D deficiency delays central nervous system myelination by inhibiting proteolipid protein trafficking from late endosome/lysosome to plasma membrane. Exp Mol Med. 2018;50(3): e457.
Zhang T, Pang W, Feng T, Guo J, Wu K, Nunez Santos M, et al. TMEM106B regulates microglial proliferation and survival in response to demyelination. Sci Adv. 2023;9(18):eadd2676.
Gallagher MD, Posavi M, Huang P, Unger TL, Berlyand Y, Gruenewald AL, et al. A Dementia-Associated Risk Variant near TMEM106B Alters Chromatin Architecture and Gene Expression. Am J Hum Genet. 2017;101(5):643–63.
Nicholson AM, Finch NA, Wojtas A, Baker MC, Perkerson RB, Castanedes-Casey M, et al. TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia. J Neurochem. 2013;126(6):781–91.
Busch JI, Unger TL, Jain N, Tyler Skrinak R, Charan RA, Chen-Plotkin AS. Increased expression of the frontotemporal dementia risk factor TMEM106B causes C9orf72-dependent alterations in lysosomes. Hum Mol Genet. 2016;25(13):2681–97.
Chen-Plotkin AS, Unger TL, Gallagher MD, Bill E, Kwong LK, Volpicelli-Daley L, et al. TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/212 cluster and affects progranulin pathways. J Neurosci. 2012;32(33):11213–27.
Suzuki H, Matsuoka M. The Lysosomal Trafficking Transmembrane Protein 106B Is Linked to Cell Death. J Biol Chem. 2016;291(41):21448–60.
Zhou X, Sun L, Brady OA, Murphy KA, Hu F. Elevated TMEM106B levels exaggerate lipofuscin accumulation and lysosomal dysfunction in aged mice with progranulin deficiency. Acta Neuropathol Commun. 2017;5(1):9.
Fedele AO, Proud CG. Chloroquine and bafilomycin A mimic lysosomal storage disorders and impair mTORC1 signalling. Biosci Rep. 2020;40(4):BSR20200905.
Bowman EJ, Graham LA, Stevens TH, Bowman BJ. The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae. J Biol Chem. 2004;279(32):33131–8.
Götzl JK, Mori K, Damme M, Fellerer K, Tahirovic S, Kleinberger G, et al. Common pathobiochemical hallmarks of progranulin-associated frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis. Acta Neuropathol. 2014;127(6):845–60.
Rhinn H, Abeliovich A. Differential Aging Analysis in Human Cerebral Cortex Identifies Variants in TMEM106B and GRN that Regulate Aging Phenotypes. Cell Syst. 2017;4(4):404-415.e5.
Colacurcio DJ, Nixon RA. Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease. Ageing Res Rev. 2016;32:75–88.
Sun Y, Li M, Zhao D, Li X, Yang C, Wang X. Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans. Elife. 2020;9:e55745.
Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, et al. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature. 2006;442(7105):916–9.
Zhou X, Paushter DH, Feng T, Sun L, Reinheckel T, Hu F. Lysosomal processing of progranulin. Mol Neurodegener. 2017;12(1):62.
Ward ME, Chen R, Huang HY, Ludwig C, Telpoukhovskaia M, Taubes A, et al. Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis. Sci Transl Med. 2017;9(385):eaah5642.
Canafoglia L, Morbin M, Scaioli V, Pareyson D, D’Incerti L, Fugnanesi V, et al. Recurrent generalized seizures, visual loss, and palinopsia as phenotypic features of neuronal ceroid lipofuscinosis due to progranulin gene mutation. Epilepsia. 2014;55(6):e56-59.
Werner G, Damme M, Schludi M, Gnörich J, Wind K, Fellerer K, et al. Loss of TMEM106B potentiates lysosomal and FTLD-like pathology in progranulin-deficient mice. EMBO Rep. 2020;21(10): e50241.
Zhou X, Brooks M, Jiang P, Koga S, Zuberi AR, Baker MC, et al. Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice. EMBO Rep. 2020;21(10): e50197.
Arrant AE, Nicholson AM, Zhou X, Rademakers R, Roberson ED. Partial Tmem106b reduction does not correct abnormalities due to progranulin haploinsufficiency. Mol Neurodegeneration. 2018;13(1):32.
Cabron AS, Borgmeyer U, Richter J, Peisker H, Gutbrod K, Dörmann P, et al. Lack of a protective effect of the Tmem106b “protective SNP” in the Grn knockout mouse model for frontotemporal lobar degeneration. Acta Neuropathol Commun. 2023;11(1):21.
Arakhamia T, Lee CE, Carlomagno Y, Duong DM, Kundinger SR, Wang K, et al. Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains. Cell. 2020;180(4):633-644.e12.
Zhou Q, Qiu H. The Mechanistic Impact of N-Glycosylation on Stability, Pharmacokinetics, and Immunogenicity of Therapeutic Proteins. J Pharm Sci. 2019;108(4):1366–77.
Brady OA, Zhou X, Hu F. Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor, TMEM106B, by Signal Peptide Peptidase-like 2a (SPPL2a). J Biol Chem. 2014;289(28):19670–80.
Yamamoto A, Lucas JJ, Hen R. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell. 2000;101(1):57–66.
Tan JX, Finkel T. A phosphoinositide signalling pathway mediates rapid lysosomal repair. Nature. 2022;609(7928):815–21.
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This study was supported by fundings from National Natural Science Foundation of China (82271471), Shanghai Municipal Science and Technology Major Project (No.2018SHZDZX01) and ZHANGJIANG LAB, Tianqiao and Chrissy Chen Institute, and the State Key Laboratory of Neurobiology and Frontiers Center for Brain Science of Ministry of Education, Fudan University.
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HSJ did the literature search, led the writing of the manuscript, devised all the figures and edited the manuscript. PY participated in the writing of the manuscript and helped to revise. JTY substantially revised and edited the manuscript. All authors read and approved the final manuscript.
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Jiao, HS., Yuan, P. & Yu, JT. TMEM106B aggregation in neurodegenerative diseases: linking genetics to function. Mol Neurodegeneration 18, 54 (2023). https://doi.org/10.1186/s13024-023-00644-1
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DOI: https://doi.org/10.1186/s13024-023-00644-1