Research ArticleNeurodegenerative Disease

A farnesyltransferase inhibitor activates lysosomes and reduces tau pathology in mice with tauopathy

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Science Translational Medicine  27 Mar 2019:
Vol. 11, Issue 485, eaat3005
DOI: 10.1126/scitranslmed.aat3005
  • Fig. 1 Baseline time course of tau pathology in the rTg4510 mouse model of tauopathy.

    (A) Representative mosaic of a coronal section from rTg4510 mouse brain immunostained with MC1 antibody (green) or GFAP (red) and counterstained with Hoechst (cell nuclei, blue). (B) The coronal section area of the micrographs was computed. At 5 weeks of age, rTg4510 transgenic (Tg) mice did not differ in brain size from their nontransgenic littermates (P = 0.942). However at 20 weeks, the coronal section area was significantly reduced in transgenic mice compared to both age-matched controls and younger transgenic animals (P = 6.94 × 10−4). (C) Twenty-week-old control littermates (Ctr) show no immunostaining of brain tissue with MC1, (D) whereas 20-week-old transgenic mice show a high density of neurons strongly labeled with MC1 in both cerebral cortex (CTX) and hippocampal formation (HPF). (E) Quantification of neurofibrillary tangles (NFTs/mm2) in the cortex and hippocampus. Representative micrographs showing microglia (Iba1-positive cells) in 20-week-old control (F) and transgenic (G) mice. (H) Quantification of microglia per square millimeter density reveals that there is no microglia age-related decline in rTg4510 transgenic mice. At 20 weeks of age in the control mouse cerebral cortex, there were 178.5 ± 66.2 microglia/mm2 versus 444.2 ± 55.9 microglia/mm2 in the rTg4510 mice (P = 1.9 × 10−3). In the control mouse hippocampus, there were 236.7 ± 17.7 microglia/mm2 versus 471.0 ± 42.0 microglia/mm2 in the rTg4510 mice (P = 5.11 × 10−3). Astrocytes labeled with GFAP antibody in (I) nontransgenic control and (J) transgenic mice at 20 weeks of age showed cortical astrogliosis accompanying MC1 immunoreactivity in the rTg4510 mice. Activated hypertrophied astrocytes are shown in the inset. (K) Percentage area in the coronal sections stained for GFAP was calculated. The cortical GFAP signal (P = 9.14 × 10−4, ANOVA) quantified in rTg4510 mice at 20 weeks of age increased compared to age-matched control mice (P = 9.2 × 10−3). The cortical GFAP signal also increased in 20-week-old transgenic mice compared to 5-week-old transgenic mice (P = 0.020). The hippocampal GFAP signal in transgenic and control mice at 5 and 20 weeks of age did not differ (P = 0.676, ANOVA). Statistics shown for Tukey’s HSD (honestly significant difference) post hoc tests. Group sizes: n = 3 (female:male ratio; control 5 wk, 2:1; Tg 5 wk, 1:2; Ctr 20 wk, 1:2; Tg 20 wk, 2:1). Scale bars, 1 mm. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Lonafarnib treatment prevents neurofibrillary tangle formation and microgliosis.

    (A) Brain coronal section area in 20-week-old rTg4510 transgenic mice that received chronic oral administration of lonafarnib (L80) versus untreated transgenic mice (Unt) or transgenic mice treated with vehicle alone (Veh). (B and C) Reduction of the extent of MC1 immunoreactivity in lonafarnib (L80)–treated transgenic mice compared to untreated mice. Scale bars, 1 mm. (D to G) Detail of insets of (B) and (C) showing representative MC1 immunoreactivity for the cortex (CTX) and hippocampus (HPF) of either untreated or lonafarnib-treated (L80) 20-week-old transgenic mice. (H) Large-scale coronal section mosaics quantified for MC1 immunoreactivity per square millimeter indicate a reduction of tau pathology after lonafarnib treatment (L80) when compared to untreated mice or animals treated with vehicle alone. (I to L) Density of microglia in the cortex and hippocampus of transgenic mice treated with lonafarnib (L80) or untreated is shown by Iba1 immunolabeling. Hippocampal microglial reactivity declined upon lonafarnib treatment. (M) Microglia quantification of coronal section mosaics in both the cortex and hippocampus of transgenic mice treated with lonafarnib. No statistically significant differences were observed in the cortex (P = 0.667, ANOVA) of lonafarnib-treated transgenic animals (489.16 ± 10.32 Iba1-positive cells/mm2) when compared to vehicle-treated (520.35 ± 10.71 Iba1-positive cells/mm2) or untreated animals (515.45 ± 11.03 Iba1-positive cells/mm2). (N to Q) Astrocytes immunostained for GFAP in the cortex or hippocampus of untreated and lonafarnib-treated rTg4510 mice and (R) quantification of GFAP signal in full coronal slices. Neither lonafarnib (L80) nor vehicle alone altered astrocytes in 20-week-old transgenic mice in the hippocampus (P = 0.236, ANOVA), but a significant reduction was observed in the cortex when comparing lonafarnib-treated to vehicle-treated mice (P = 0.042). Statistics shown for Tukey’s HSD post hoc tests. Group sizes: n = 6 (female:male ratio; Unt, 1:5; Veh, 3:3; L80, 3:3). Scale bars, 100 μm. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Chronic lonafarnib treatment ameliorates a nest building deficit in rTg4510 mice.

    (A) At 20 weeks of age, littermate control mice display normal nest shredding behavior, but (B) transgenic animals failed to demonstrate nest shredding, as shown in the representative photographs. (C) Lonafarnib-treated 20-week-old rTg4510 animals shredded their nest. (D) Twenty-week-old transgenic mice had nest quality scores averaging 0.3 ± 0.1 in untreated mice or 0.2 ± 0.1 in mice treated with vehicle (Veh) alone, whereas the age-matched WT mouse nest shredding score averaged 3.7 ± 0.1 (both, P < 5.3 × 10−9). Chronic and intermittent oral administration of lonafarnib (L80) (80 mg/kg per day) rescued nest building, with nest scores averaging 3.5 ± 0.2 for treated mice. Qualitative scores were blindly assigned by observers using a scale from zero for untouched nesting material to five for a fully shredded nest (n = 6). (E) Twenty-week-old littermate control mice buried 80 ± 2.0% of the 20 marbles in 30 min, but (F) 20-week-old transgenic mice completely lacked digging behavior. (G) Control mice increased the percentage of marbles buried with age. They buried 40% of the marbles at 5 weeks of age and peaked at 30 weeks of age with an average of 80% of the marbles buried. Transgenic mice failed to bury marbles as early as 5 weeks of age {two-way ANOVA: genotype, P < 2 × 10−16; age, P = 0.140 [not significant (n.s.)]; interaction, P = 1.93 × 10−5}. (H) Marble burial deficits were rescued by neither lonafarnib (L80) nor vehicle treatment. Data are presented as box plots of percentage marbles buried per treatment group. Statistics shown for post hoc Wilcoxon tests. n = 9 (female:male ratio; Ctr Unt, 8:4; Tg Unt, 4:8; Tg Veh, 5:4; Tg L80, 4:5). ***P < 0.001.

  • Fig. 4 Inhibition of farnesyltransferase activates autophagy.

    (A to C) NIH3T3 cells expressing the tandem reporter mCherry-GFP-LC3B were exposed to the indicated concentrations of lonafarnib for 48 hours. The quantified number of autophagic vacuoles (AV), autophagosomes (APG), and autolysosomes (AUT) is shown. (D) NIH3T3 cells expressing the KFERQ-Dendra reporter were photoswitched and treated with lonafarnib as indicated above. CMA was quantified by the number of fluorescent puncta positive for the photoconverted Dendra per cell. (E) NIH3T3 cells expressing N- and C-terminal KFERQ-split-Venus and treated with lonafarnib for 48 hours either were treated with 20 mM NH4Cl and 100 μM leupeptin (N/L) or were left untreated for the last 24 hours to quantify the effect of lonafarnib on targeting (None) and degradation (N/L) by endosomal microautophagy. Quantifications in (A) to (G) were done in at least 2500 cells per condition in three different experiments using high-content microscopy. (F) Results for NIH3T3 cells expressing degron-GFP and treated with lonafarnib for 48 hours and supplemented with 100 μM lactacystin for the last 12 or 24 hours. Proteasome-dependent degradation was calculated as the increase in the intensity of fluorescence upon lactacystin addition and after discounting the increase observed in cells treated under the same conditions but expressing a nonubiquitinable degron-GFP mutant. Values are expressed relative to the proteasome degradation in cells not treated with lonafarnib that were given an arbitrary value of one. (G) Total rates of intracellular protein degradation measured in NIH3T3 cells labeled with [3H]leucine for 48 hours. The rate of proteolysis was calculated as the percentage of the initial acid-precipitable radioactivity (proteins) transformed into acid-soluble radioactivity (amino acids and small peptides) at the indicated times. (H) The contribution of lysosomes to total protein degradation was analyzed by supplementing cells with NH4Cl and leupeptin. Data are presented as means ± SEM (n = 6 wells in three independent experiments). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 Rhes suppression mimics the protective effect of lonafarnib in rTg4510 mice.

    Ten-week-old transgenic rTg4510 mice were intracranially infected with AAV 2/5 containing either U6, Rhes-miR, or Rhes-WT constructs and were sacrificed at 20 weeks of age. (A to C) Full mosaic immunohistochemistry for MC1 immunoreactivity after Rhes suppression by Rhes-miR showed fewer neurofibrillary tangles than did transduction with U6 or Rhes-WT. (D to F) Full mosaic immunohistochemistry for Iba1 was also reduced in mice treated with Rhes-miR. The quantification of micrographs indicates (G) significantly increased coronal section area for Rhes-miR (40.53 ± 0.70 mm2; P = 0.002) but not Rhes-WT (36.41 ± 0.79 mm2; P = 0.307) in injected transgenic mice compared to U6 (37.33 ± 0.36 mm2). (H) There were a reduced number of MC1-positive cells per square millimeter both in the cerebral cortex (CTX) and hippocampus (HPF). The cortical density of MC1-positive cells per square millimeter in animals injected with U6 was 157.81 ± 12.56 compared to Rhes-miR (78.76 ± 8.88 MC1-positive cells/mm2; P = 3.90 × 10−4) or Rhes overexpression (172.15 ± 13.12 MC1-positive cells/mm2; P = 0.487). In the hippocampus, injected mice had 86.09 ± 9.28 MC1-positive cells/mm2 compared to Rhes-miR (49.15 ± 7.01 MC1-positive cells/mm2; P = 3.61 × 10−3) or Rhes overexpression (92.86 ± 9.64 MC1-positive cells/mm2; P = 0.635). (I) Rhes-miR reduced the number of microglia per square millimeter in the cortex and hippocampus in Rhes-miR–injected rTg4510 mice. Rhes-miR–injected transgenic mice showed a significant reduction in the cortex (compared to U6 control; P = 1.50 × 10−4) and hippocampus (compared to U6 control; P = 3.02 × 10−4), whereas Rhes-WT showed a reduction in the cortex (P = 0.027) with no effect observed in the hippocampus (P = 0.531). (J) No significant change was observed in astrocytic immunoreactivity in the cerebral cortex for either Rhes-miR (P = 0.182) or Rhes-WT overexpression (P = 0.094) compared to U6 controls. In the hippocampus, Rhes-miR significantly reduced GFAP immunostaining compared to U6 controls (P = 0.029) but not when compared to Rhes-WT overexpression (P = 0.343). n = 3 (female:male ratio; U6, 0:3; Rhes-miR, 0:3; Rhes, 3:0). Scale bars, 1 mm. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Farnesyltransferase inhibition prevents Rhes-mediated tau accumulation and activates autophagy.

    (A) Overexpression of either Rhes-WT or the GTPase-inactive mutant Rhes-S33N but not Rhes silenced by microRNA (Rhes-miR) in hippocampal primary mouse neurons increased PHF-1 phospho-tau. Representative blot (A) and densitometry quantification (B) of independent replicates. (C) Farnesyltransferase inhibition with lonafarnib rescued the Rhes-induced increase in phosphorylated tau in a dose-dependent manner. Representative blot (C) and densitometry quantification (D) of independent replicates. β-Actin was used for a loading and normalization control. A four-parameter log-logistic model fit (R package, drc) is shown [(B) P = 0.006; (C) P = 0.010; (D) P < 1 × 10−4, IC50 = 61.00 nM, P < 1 × 10−4]. (E to H) Primary mouse neuronal cultures (Mock) were transduced with AAV 2/5 to overexpress Rhes and then were either left untreated (Rhes-positive), treated with 250 nM lonafarnib alone (Lon), or additionally treated either with a cocktail containing 20 mM NH4Cl and 100 μM leupeptin (N/L) to block lysosomal-mediated proteolysis or with 5 μM MG-132 (N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal) (MG) to block proteasome activity. Cell lysates were obtained at 12 hours, or cell media and inhibitors were replenished at 12 hours and lysates were collected at 24 hours, as indicated. (E) Proteins in the cell lysates were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and then Western-blotted for PHF-1 S496/phospho-tau, total tau (TAU5), and p62. (F) PHF-1 antibody was normalized to TAU5; (G) TAU5 or (H) p62 was normalized to β-tubulin III and plotted as means ± SEM. Lonafarnib prevented a Rhes-mediated PHF-1 increase, an effect reversed by lysosomal proteolysis inhibition (N/L) but not by proteasome inhibition (MG). (I) Western blot of cortical lysates from untreated rTg4510 mice at 20 weeks of age (Ctr), untreated (Unt) transgenic mice at 5 weeks of age, and either vehicle (Veh) or lonafarnib-treated transgenic mice at 20 weeks of age. Western blots (I) were treated with antibodies to pathological tau, total tau (TAU5), and β-tubulin III as the loading control; (J to N) their respective densitometric quantifications are presented. Phosphorylated and conformational tau were normalized to TAU5, and TAU5 was normalized to β-tubulin III (n = 3). (O) Sarkosyl-insoluble tau fractions were isolated from mouse cortical lysates; a 64-kDa apparent high–molecular weight tau band is indicated by the asterisk in cortical lysates from aged rTg4510 mice, but no comparable band was detectable in control, young, or age-matched lonafarnib-treated rTg4510 mice. (A and B and I to N) n = 3. (I to N) Female:male ratio; Unt Ctr 20 wk, 0:3; Unt Tg 5 wk, 1:2; Veh Tg 20 wk, 1:2; L80 Tg 20 wk, 2:1. (C and D and E to H) N = 5 independent experiments. Statistical analysis: one-way ANOVA and Tukey’s HSD post hoc tests.*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 1 × 10−4.

  • Fig. 7 RASD2 expression is reduced in transcriptome profiles of hiPSC-derived neurons from patients with frontotemporal dementia carrying MAPT mutations.

    (A) RNAseq of hiPSC-derived neurons from patients with frontotemporal dementia carrying MAPT mutations (G55R, P301L, V337M, and R406W) or a C9ORF72 expansion versus three age-matched healthy controls cultured for 5 weeks. Box plots of normalized CPMs per library are shown. (B) RASD2 expression was deregulated across each patient line carrying MAPT mutations. FDR, false discovery rate. (C) MAPT expression did not change across iPSC-derived neurons with different MAPT mutations. (D) TUBB3 was highly expressed and remained unaltered across samples. Digital PCR data (E to G) presented as mean copies/μl ± SEM validated the findings of each of these genes for each cell line studied. RASD2 suppression in the presence of tau mutations in neurons differentiated from hiPSCs compared to their respective isogenic control lines was verified by TaqMan RT-PCR using (H) F0510 cells harboring P301L and P301S mutations (P = 1.45 × 10−3, ANOVA), (I) hiPSC-derived neurons harboring the MAPT-V337M mutation (P = 0.035, t test), and (J) hiPSC-derived neurons harboring the MAPT-R406W mutation (P = 2.14 × 10−3, t test). (K to M) MAPT expression remained unchanged in hiPSC-derived neurons carrying different MAPT mutations and in the corrected isogenic lines. *P < 0.05 and **P < 0.01.

  • Fig. 8 Rhes expression increases as neurons age, which can be prevented by tau mutations.

    (A) Rhes expression is reduced in MAPT-P301L and MAPT-V337M hiPSC-derived neurons during hiPSC differentiation into neurons as early as the neurorosette stage; Rhes expression increases during differentiation into neurons of hiPSCs carrying MAPT-WT. (B) MAPT expression increases continuously during neuronal differentiation regardless of MAPT genotype. (C) Rhes quantification in the brains of rTg4510 transgenic mice and littermate controls. Rhes increases as both transgenic and control mice age, but these increases are significantly lower than for younger transgenic mice [two-way ANOVA: age, P = 3.24 × 10−3; genotype, P = 8.01 × 10−3; interaction, P = 0.88 (n.s.)]. (D to M) Rhes reduction in 20-week-old transgenic mice by both lonafarnib and Rhes-miR treatments. Untreated rTg4510 mice had a mean of 693.3 ± 27.0 Rhes-positive cells/mm2 in the cortex (CTX) and 530.0 ± 54.9 cells/mm2 in the hippocampus (HPF). Chronic administration of lonafarnib reduced Rhes to 409.1 ± 18.5 cells/mm2 (P = 5.62 × 10−3) in the cortex and to 252.6 ± 23.7 cells/mm2 (P = 9.29 × 10−3) in the hippocampus. Rhes-miR treatment reduced Rhes to 396.27 ± 31.52 cells/mm2 (P = 0.002) in the cortex and to 268.1 ± 16.1 cells/mm2 (P = 0.033) in the hippocampus. Statistics shown for Wilcoxon tests. (A to C) n = 3 (female:male ratio; Ctrs 5 wk 1:2, 10 wk 0:3, 20 wk 2:1, 32 wk 2:1; Tg 5 wk 3:0, 10 wk 0:3, 20 wk 2:1, 32 wk 1:2). (D to M) n = 3 (female:male ratio; Unt, 0:3; L80, 3:3; Veh, 3:3; Rhes-miR, 1:2). Scale bars, 100 μm. *P < 0.05 and **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/485/eaat3005/DC1

    Materials and Methods

    Fig. S1. Tau pathology progression in rTg4510 transgenic mice.

    Fig. S2. PHF-1 immunoreactivity is reduced by lonafarnib treatment.

    Fig. S3. Acute treatment of lonafarnib does not alter tau pathology in aged rTg4510 transgenic mice.

    Fig. S4. Effect of lonafarnib in macroautophagy and CMA.

    Fig. S5. Farnesyltransferase activity inhibition activates autophagy.

    Fig. S6. Sumo and ubiquitin are reduced by lonafarnib and Rhes-miR treatments.

    Fig. S7. Lonafarnib’s effect on Rhes localization.

    Fig. S8. Degradation of Rhes is insensitive to lysosomal proteolysis blocking.

    Fig. S9. Lonafarnib treatment is effective in reducing phospho-tau in iPSC-derived neurons.

    Fig. S10. Transcriptomic analysis of hiPSC-derived neurons harboring tau mutations.

    Fig. S11. Ubiquitinated and sumoylated tau are not altered by lonafarnib treatment.

    Fig. S12. Model for a Rhes pathway mechanism.

    Movie S1. Lonafarnib attenuates behavioral circling in rTg4510 (mp4).

    Data file S1: Differentially expressed genes in hiPSC-derived neurons with MAPT variants (Excel file).

    Data file S2. Quantification data for rTg4510-treated mice (excel file).

    References (6165)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Tau pathology progression in rTg4510 transgenic mice.
    • Fig. S2. PHF-1 immunoreactivity is reduced by lonafarnib treatment.
    • Fig. S3. Acute treatment of lonafarnib does not alter tau pathology in aged rTg4510 transgenic mice.
    • Fig. S4. Effect of lonafarnib in macroautophagy and CMA.
    • Fig. S5. Farnesyltransferase activity inhibition activates autophagy.
    • Fig. S6. Sumo and ubiquitin are reduced by lonafarnib and Rhes-miR treatments.
    • Fig. S7. Lonafarnib’s effect on Rhes localization.
    • Fig. S8. Degradation of Rhes is insensitive to lysosomal proteolysis blocking.
    • Fig. S9. Lonafarnib treatment is effective in reducing phospho-tau in iPSC-derived neurons.
    • Fig. S10. Transcriptomic analysis of hiPSC-derived neurons harboring tau mutations.
    • Fig. S11. Ubiquitinated and sumoylated tau are not altered by lonafarnib treatment.
    • Fig. S12. Model for a Rhes pathway mechanism.
    • References (6165
    )

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1. Lonafarnib attenuates behavioral circling in rTg4510 (mp4).
    • Data file S1. Differentially expressed genes in hiPSC-derived neurons with MAPT variants (Excel file).
    • Data file S2. Quantification data for rTg4510-treated mice (excel file).

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