Loss of TMEM106B potentiates lysosomal and FTLD-like pathology in progranulin-deficient mice.

Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP-43 pathology in single and double knockout animals. Grn-/- /Tmem106b-/- mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease-associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn-/- knockouts only occasionally show TDP-43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP-43. Thus, a loss of function of TMEM106B may enhance the risk for GRN-associated FTLD by reduced protein turnover in the lysosomal/autophagic system.

Progranulin transcripts with short and long 5' untranslated regions (UTRs) are differentially expressed via posttranscriptional and translational repression.

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency.

Common pathobiochemical hallmarks of progranulin-associated frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis.

Heterozygous loss-of-function mutations in the progranulin (GRN) gene and the resulting reduction of GRN levels is a common genetic cause for frontotemporal lobar degeneration (FTLD) with accumulation of TAR DNA-binding protein (TDP)-43. Recently, it has been shown that a complete GRN deficiency due to a homozygous GRN loss-of-function mutation causes neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. These findings suggest that lysosomal dysfunction may also contribute to some extent to FTLD. Indeed, Grn(-/-) mice recapitulate not only pathobiochemical features of GRN-associated FTLD-TDP (FTLD-TDP/GRN), but also those which are characteristic for NCL and lysosomal impairment. In Grn(-/-) mice the lysosomal proteins cathepsin D (CTSD), LAMP (lysosomal-associated membrane protein) 1 and the NCL storage components saposin D and subunit c of mitochondrial ATP synthase (SCMAS) were all found to be elevated. Moreover, these mice display increased levels of transmembrane protein (TMEM) 106B, a lysosomal protein known as a risk factor for FTLD-TDP pathology. In line with a potential pathological overlap of FTLD and NCL, Ctsd(-/-) mice, a model for NCL, show elevated levels of the FTLD-associated proteins GRN and TMEM106B. In addition, pathologically phosphorylated TDP-43 occurs in Ctsd(-/-) mice to a similar extent as in Grn(-/-) mice. Consistent with these findings, some NCL patients accumulate pathologically phosphorylated TDP-43 within their brains. Based on these observations, we searched for pathological marker proteins, which are characteristic for NCL or lysosomal impairment in brains of FTLD-TDP/GRN patients. Strikingly, saposin D, SCMAS as well as the lysosomal proteins CTSD and LAMP1/2 are all elevated in patients with FTLD-TDP/GRN. Thus, our findings suggest that lysosomal storage disorders and GRN-associated FTLD may share common features.

Membrane orientation and subcellular localization of transmembrane protein 106B (TMEM106B), a major risk factor for frontotemporal lobar degeneration.

TMEM106B was identified as a major risk factor in a genome-wide association study for frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP)-43 pathology. The most significant association of TMEM106B single nucleotide polymorphisms with risk of FTLD-TDP was observed in patients with progranulin (GRN) mutations. Subsequent studies suggested an inverse correlation between TMEM106B expression and GRN levels in patient serum. However, in this study, this was not confirmed as we failed to detect a significant alteration of GRN levels upon knockdown or exogenous expression of TMEM106B in heterologous cells. To provide a basis for understanding TMEM106B function in health and disease, we investigated the membrane orientation and subcellular localization of this completely uncharacterized protein. By differential membrane extraction and sequential mutagenesis of potential N-glycosylation sites, we identified TMEM106B as a type 2 integral membrane protein with a highly glycosylated luminal domain. Glycosylation is partially required for the transport of TMEM106B beyond the endoplasmic reticulum to late cellular compartments. Endogenous as well as overexpressed TMEM106B localizes to late endosomes and lysosomes. Interestingly, the inhibition of vacuolar H(+)-ATPases significantly increased the levels of TMEM106B, a finding that may provide an unexpected biochemical link to GRN, because this protein is also strongly increased under the same conditions. Our findings provide a biochemical and cell biological basis for the understanding of the pathological role of TMEM106B in FTLD, an incurable neurodegenerative disorder.

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism.

Microglia adopt numerous fates with homeostatic microglia (HM) and a microglial neurodegenerative phenotype (MGnD) representing two opposite ends. A number of variants in genes selectively expressed in microglia are associated with an increased risk for neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Among these genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). Both cause neurodegeneration by mechanisms involving loss of function. We have now isolated microglia from Grn-/- mice and compared their transcriptomes to those of Trem2-/-mice Surprisingly, while loss of Trem2 enhances the expression of genes associated with a homeostatic state, microglia derived from Grn-/- mice showed a reciprocal activation of the MGnD molecular signature and suppression of gene characteristic for HM The opposite mRNA expression profiles are associated with divergent functional phenotypes. Although loss of TREM2 and progranulin resulted in opposite activation states and functional phenotypes of microglia, FDG (fluoro-2-deoxy-d-glucose)-µPET of brain revealed reduced glucose metabolism in both conditions, suggesting that opposite microglial phenotypes result in similar wide spread brain dysfunction.

Early increase of CSF sTREM2 in Alzheimer's disease is associated with tau related-neurodegeneration but not with amyloid-ß pathology.

BACKGROUND: TREM2 is a transmembrane receptor that is predominantly expressed by microglia in the central nervous system. Rare variants in the TREM2 gene increase the risk for late-onset Alzheimer's disease (AD). Soluble TREM2 (sTREM2) resulting from shedding of the TREM2 ectodomain can be detected in the cerebrospinal fluid (CSF) and is a surrogate measure of TREM2-mediated microglia function. CSF sTREM2 has been previously reported to increase at different clinical stages of AD, however, alterations in relation to Amyloid ß-peptide (Aß) deposition or additional pathological processes in the amyloid cascade (such as tau pathology or neurodegeneration) remain unclear. In the current cross-sectional study, we employed the biomarker-based classification framework recently proposed by the NIA-AA consensus guidelines, in combination with clinical staging, in order to examine the CSF sTREM2 alterations at early asymptomatic and symptomatic stages of AD. METHODS: A cross-sectional study of 1027 participants of the Alzheimer's Disease Imaging Initiative (ADNI) cohort, including 43 subjects carrying TREM2 rare genetic variants, was conducted to measure CSF sTREM2 using a previously validated enzyme-linked immunosorbent assay (ELISA). ADNI participants were classified following the A/T/N framework, which we implemented based on the CSF levels of Aß1-42 (A), phosphorylated tau (T) and total tau as a marker of neurodegeneration (N), at different clinical stages defined by the clinical dementia rating (CDR) score. RESULTS: CSF sTREM2 differed between TREM2 variants, whereas the p.R47H variant had higher CSF sTREM2, p.L211P had lower CSF sTREM2 than non-carriers. We found that CSF sTREM2 increased in early symptomatic stages of late-onset AD but, unexpectedly, we observed decreased CSF sTREM2 levels at the earliest asymptomatic phase when only abnormal Aß pathology (A+) but no tau pathology or neurodegeneration (TN-), is present. CONCLUSIONS: Aß pathology (A) and tau pathology/neurodegeneration (TN) have differing associations with CSF sTREM2. While tau-related neurodegeneration is associated with an increase in CSF sTREM2, Aß pathology in the absence of downstream tau-related neurodegeneration is associated with a decrease in CSF sTREM2.

Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice.

BACKGROUND: Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear. METHODS: mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins. RESULTS: We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN. CONCLUSIONS: The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells.

CSF progranulin increases in the course of Alzheimer's disease and is associated with sTREM2, neurodegeneration and cognitive decline.

Progranulin (PGRN) is predominantly expressed by microglia in the brain, and genetic and experimental evidence suggests a critical role in Alzheimer's disease (AD). We asked whether PGRN expression is changed in a disease severity-specific manner in AD We measured PGRN in cerebrospinal fluid (CSF) in two of the best-characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). In carriers of AD causing dominant mutations, cross-sectionally assessed CSF PGRN increased over the course of the disease and significantly differed from non-carriers 10 years before the expected symptom onset. In late-onset AD, higher CSF PGRN was associated with more advanced disease stages and cognitive impairment. Higher CSF PGRN was associated with higher CSF soluble TREM2 (triggering receptor expressed on myeloid cells 2) only when there was underlying pathology, but not in controls. In conclusion, we demonstrate that, although CSF PGRN is not a diagnostic biomarker for AD, it may together with sTREM2 reflect microglial activation during the disease.

Reduced secretion and altered proteolytic processing caused by missense mutations in progranulin.

Progranulin (GRN) is a secreted growth factor involved in various cellular functions, and loss-of-function mutations are a major cause of frontotemporal lobar degeneration (FTLD) with TDP-43 positive pathology. Most FTLD-related GRN mutations are nonsense mutations resulting in reduced GRN expression. Nonsynonymous GRN missense mutations have been described as risk factor for neurodegenerative brain diseases, but their pathogenic nature remains largely elusive. We identified a double missense mutation in GRN leading to amino acid changes p.D33E and p.G35R in an FTLD patient from Turkish origin. Biochemical and cell biological analysis of the double-mutation together with 2 so-far uncharacterized GRN missense mutations (p.C105R and p.V514M) revealed a reduced secretion efficiency of the GRN p.D33E/p.G35R and p.C105R proteins. Furthermore, loss of the conserved cysteine residue affects protein folding and altered proteolytic processing by neutrophil elastase and proteinase 3. Our data indicate that the described variants may cause a loss-of-function, albeit to a lesser extent than GRN null mutations, and hence could be considered as low-penetrant risk factors for neurodegenerative diseases.

Missense mutations in the progranulin gene linked to frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions reduce progranulin production and secretion.

Loss of function mutations in progranulin cause tau-negative frontotemporal lobar degeneration with ubiquitin-positive inclusions. A major protein component of these inclusions is TDP-43, which becomes hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments, which apparently translocate from nuclei to the cytoplasm. Most progranulin mutations are nonsense mutations resulting in nonsense-mediated mRNA decay and consequently reduced progranulin protein levels. However, some missense mutations are described that occur within the signal sequence and mature progranulin. We now demonstrate that a progranulin mutation located within the signal sequence (PGRN A9D) results in cytoplasmic missorting with extremely low expression. In contrast, two other progranulin mutations (PGRN P248L and R432C) are expressed as immature proteins but are inefficiently transported through and partially degraded within the secretory pathway, resulting in a significantly reduced secretion. Thus apparently all progranulin mutations cause reduced protein expression or secretion, although by different cellular mechanisms. To investigate a putative relationship between reduced expression of progranulin and TDP-43 relocalization and deposition, we down-regulated progranulin in human cell lines and in zebrafish. Upon reduction of progranulin, neither a major redistribution of TDP-43 nor proteolytic processing to disease-characterizing C-terminal fragments could be observed.

A basolateral sorting signal directs ADAM10 to adherens junctions and is required for its function in cell migration.

ADAM10 (a disintegrin and metalloprotease) initiates regulated intramembrane proteolysis by shedding the ectodomain of a number of different substrates. Shedding is followed by subsequent intramembrane proteolysis leading to the liberation of intracellular domains capable of nuclear signaling. ADAM10 substrates have been found at cell-cell contacts and are apparently involved in cell-cell interaction and cell migration. Here we have investigated the cellular mechanism that guides ADAM10 to substrates at cell-cell contacts. We demonstrate that intracellular trafficking of ADAM10 critically requires a novel sorting signal within its cytoplasmic domain. Sequential deletion of the cytoplasmic domain and site-directed mutagenesis suggest that a potential Src homology 3-binding domain is essential for ADAM10 sorting. In a polarized epithelial cell line this motif not only targets ADAM10 to adherens junctions but is also strictly required for ADAM10 function in E-cadherin processing and cell migration.