Elevated TMEM106B levels exaggerate lipofuscin accumulation and lysosomal dysfunction in aged mice with progranulin deficiency

Abstract

Mutations resulting in haploinsufficiency of progranulin (PGRN) cause frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), a devastating neurodegenerative disease. Accumulating evidence suggest a crucial role of progranulin in maintaining proper lysosomal function during aging. TMEM106B has been identified as a risk factor for frontotemporal lobar degeneration with progranulin mutations and elevated mRNA and protein levels of TMEM106B are associated with increased risk for frontotemporal lobar degeneration. Increased levels of TMEM106B alter lysosomal morphology and interfere with lysosomal degradation. However, how progranulin and TMEM106B interact to regulate lysosomal function and frontotemporal lobar degeneration (FTLD) disease progression is still unclear. Here we report that progranulin deficiency leads to increased TMEM106B protein levels in the mouse cortex with aging. To mimic elevated levels of TMEM106B in frontotemporal lobar degeneration (FTLD) cases, we generated transgenic mice expressing TMEM106B under the neuronal specific promoter, CamKII. Surprisingly, we found that the total protein levels of TMEM106B are not altered despite the expression of the TMEM106B transgene at mRNA and protein levels, suggesting a tight regulation of TMEM106B protein levels in the mouse brain. However, progranulin deficiency results in accumulation of TMEM106B protein from the transgene expression during aging, which is accompanied by exaggerated lysosomal abnormalities and increased lipofuscin accumulation. In summary, our mouse model nicely recapitulates the interaction between progranulin and TMEM106B in human patients and supports a critical role of lysosomal dysfunction in the frontotemporal lobar degeneration (FTLD) disease progression.

Keywords: Frontotemporal lobar degeneration (FTLD); Lipofuscin; Lysosome; Progranulin; TMEM106B.

Fig. 1

Generation of TMEM106B transgenic mice. a Schematic drawing of the expression construct used to inject the pronuclei of fertilized eggs. Human TMEM106B and poly A sequence were cloned into the NotI site of pMM403 and the cassette was excised with SfiI for injection. b qPCR analysis of mouse TMEM106B and human TMEM106B mRNA levels in the offspring of highly expressed transgenic line and WT littermate controls of 4–5 months of age. Relative mRNA levels are normalized to actin. n = 3, student’s t-test, *, p < 0.01. c Mass spectrometry analysis of TMEM106B from the transgenic line. TMEM106B protein was immunoprecipitated from the cortical lysates of the transgenic line and the IP product was trypsin digested and subject to mass spec analysis

Fig. 2

TMEM106B protein levels are tightly regulated in the mouse cortex. (a, b) TMEM106B protein levels are not changed in the transgenic mice of 4–5 months of age (a) or 17-20months of age (b). Cortical lysates from the transgenic and littermate controls were subjected to Western blot with anti- TMEM106B antibodies. n = 3, student’s t-test. ns, no significance

Fig. 3

Regulation of TMEM106B protein levels by PGRN in aged brain. a Endogenous TMEM106B protein levels are elevated in PGRN−/− mice but not in wild type mice upon aging. Cortical lysates from mice of indicated genotypes and ages (young: 4–5 months; aged: 17–20 months old) were subjected to Western blot with anti- TMEM106B antibodies. b Quantification of (a) to compare TMEM106B protein levels between young and aged mice in the WT (left) or GRN−/− (right) background. c Quantification of TMEM106B protein levels between WT and GRN−/− mice during young or aged conditions. n = 3-5, student’s t-test. *, p < 0.05; **, p < 0.01. ns, no significance

Fig. 4

Expression of the TMEM106B transgene results in elevated TMEM106B levels in aged PGRN−/− mice. a Transgene expression does not lead to changes in TMEM106B protein levels in young PGRN−/− mice of 4–5 months old compared to littermate WT controls. Cortical lysates from the transgenic and littermate controls were subjected to Western blot with anti- TMEM106B antibodies. b TMEM106B transgene expression leads to increased TMEM106B protein levels in aged PGRN−/− mice (17-20 months old). n = 3, student’s t-test. *, p < 0.05; ns, no significance

Fig. 5

TMEM106B transgene expression increases lipofuscin accumulation in PGRN deficient mice. a TMEM106B transgene expression results in increased autofluorescence in the cortex and thalamus in PGRN-/- mice but has no effect on WT mice. Brain sections from 17-20 months old mice of indicated genotypes were imaged at 594nm for auto fluorescent signals (red). The auto fluorescent signals were quantified by Image J. Hochest 33324 was used as a marker for nuclei (blue). Scale bar=100 μm (b, c) Quantification of (a). n=3, student’s t-test, **, p<0.01; ***, p<0.001; ns, no significance

Fig. 6

TMEM106B transgene expression increases SCMAS aggregation in PGRN deficient mice. Brain sections (a, cortex; b, thalamus) from 17-20months old mice of indicated genotypes were stained with anti-SCMAS and anti-cathepsin D antibodies. Scale bar = 40 μm

Fig. 7

TMEM106B transgene expression exacerbates lysosomal abnormalities in PGRN deficient mice. a TMEM106B transgene expression results in more lysosomal enlargement in PGRN−/− mice but has no effect on WT mice. Brain sections from 17-20 month old mice of indicated genotypes were stained with anti-TMEM106B, anti-LAMP1 and anti-cathepsin D antibodies. Neurons from layer III-V cortical regions were shown as examples. Scale bar = 20 μm (inset: 2 μm) (b) Percentage of neurons containing enlarged lysosomes (>1 μm) were quantified for experiment in (a). c Percentage of neurons containing more than 10 enlarged lysosomes (>1 μm) were quantified for experiment in (a). n = 3, one-way ANOVA, *, p < 0.05; ***, p < 0.001; ns, no significance

Fig. 8

A model for the cross-regulation between TMEM106B and PGRN in the transgenic mouse model and in FTLD/PGRN patients with TMEM106B risk allele. Lysosomal dysfunction caused by PGRN deficiency during aging leads to TMEM106B accumulation, which results in more severe lysosomal pathology and eventually neuronal death in FTLD. PGRN might also play a direct role in regulating TMEM106B levels (dashed line)