Research ArticleNEUROSCIENCE

Persistent repression of tau in the brain using engineered zinc finger protein transcription factors

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Science Advances  19 Mar 2021:
Vol. 7, no. 12, eabe1611
DOI: 10.1126/sciadv.abe1611
  • Fig. 1 Tau-targeted ZFP-TFs reduce mouse tau mRNA and protein expression in vitro.

    (A) Principle of ZFP-TF target DNA sequence recognition. Engineered arrays of ZFPs recognizing specific DNA triplets bind a specific genomic DNA sequence. Fusion to a TF enables repression of target gene transcription. (B) The binding locations of the lead ZFP-TF.89 downstream of the mouse MAPT gene TSS in the mouse chromosome 11 ZFP-TF.89. Gray bar indicates the location of the MAPT gene. Blue arrow indicates the coding direction of the ZFP-TF.89. (C) Adeno-associated virus serotype 9 (AAV9) constructs with CMV (ubiquitous) and hSyn1 (neuron-specific) promoters for expression of nuclear ZFP-TF.89v (N-terminal SV40 nuclear localization sequence on ZFPs) and cytoplasmic yellow/green fluorescent protein Venus as a transduction marker, separated by a self-cleaving 2a peptide. (D) Expression of AAV ZFP-TF.89v under CMV and hSyn1 promoter in primary cortical mouse neurons (DIV 7; 4 days p.i.). hSyn1-driven 89v expression shows higher tau mRNA repression (pink bars) at the same viral doses (MOI). ZFP-TF.72v, a control ZFP-TF with ZFP array without binding sequence in the mouse genome, shows no tau repression (white bars). Data are presented as means ± SD, n = 3 experiments, and data are normalized to tau mRNA in ZFP-TF.72v–expressing neurons at the highest dose (MOI = 3 × 105). (E) mRNA array data (volcano plots) show small changes in gene expression—other than MAPT—for CMV.89 or PBS compared to CMV.72v-treated primary neurons. Other significantly (>2-fold change, P < 0.01) down-regulated (red) and up-regulated (green) genes are listed. RNA array data are available in table S3. Data are presented as means ± SEM. One-way ANOVA with Sidak’s test, n = 5 to 6 biological replicates.

  • Fig. 2 Tau-targeted ZFP-TFs reduce tau mRNA and protein expression in vivo.

    (A) Location of hippocampal injection sites and subsequent ZFP-TF.89v expression in the mouse brain. The left hemisphere received two 1.5-μl injections of AAV9 CMV.89v (CMV.72v) into the anterior and posterior hippocampus, and the right hemisphere received two injections of PBS of the same volume into the same coordinates. Some hippocampi did not receive an injection and served as noninjected (NI) controls. Representative images of coronal brain sections show the widespread expression of the Venus reporter protein in the left hippocampus. Venus labeling in the right hemisphere results from contralateral projections of CMV.89v-transduced neurons in the left hippocampus. (B) mRNA levels of ZFP-TF (ZFP; green) and tau (pink) in CMV.89v- and hSyn1.89v-injected hippocampi. After 6 weeks, CMV.89v reduces tau mRNA down to 12% (by 88%) and hSyn1.89v down to 36% (by 64%), whereas control CMV.72v- or hSyn1.72v-injected hippocampi have the same amount of tau mRNA as noninjected and PBS-injected mice. After 11 months, tau was still reduced by 80% in CMV.89v hippocampi. Data are presented as means ± SEM. One-way ANOVA with Sidak’s test for multiple comparisons, reference for significance indicated by arrowhead, n = 3 mice per group. (C) Tau protein reduction in hippocampal extracts of CMV.89v- and hSyn1.89v-injected animals demonstrated by Western blot of lysates 6 weeks p.i. CMV.89v reduced total tau protein expression by ~74% and hSyn1.89v by ~82%. CMV.72v, noninjected, and PBS-injected hippocampi had similar tau protein levels. One-way ANOVA with Sidak’s posttest, n = 3 mice per group.

  • Fig. 3 Effective tau mRNA repression in individual ZFP-TF–expressing neurons.

    (A) Coronal brain sections from hSyn1.89v-injected mice immunolabeled for Venus (green) and tau protein (gray) and hybridized with fluorescently labeled mouse tau-specific RNA probe (pink). Already after 2 weeks, hSyn1.89v-injected hippocampi show a pronounced reduction in neuronal tau mRNA signal in the dentate gyrus (DG) and CA1 compared to hSyn1.72v-expressing hippocampi. (B) Individual neuronal cell bodies (=soma including nucleus) in CA2 were analyzed for tau mRNA foci. (C) Number of tau mRNA foci per cell body in CA2 and CA3: hSyn1.72v-injected mice: 18 to 24 foci per cell body, with 21.6 ± 1.8 (means ± SEM) foci across mice; hSyn1.89v-injected mice: 9 to 13 foci per cell body with 10.6 ± 1.1 foci per cell body across mice. Data are presented as means ± SEM for each mouse (M1 to M3) and each group, n = 60 to 215 neurons per mouse, three animals per group. Two-tailed Student’s t test. (D) Two-dimensional histogram of the distribution of tau mRNA foci per Venus+ neuron (y axis) and the corresponding neuronal Venus intensity (x axis). The most probable number (from Gaussian fit to distribution; means ± SD) of foci is ~20 in hSyn1.72v- and ~3 in hSyn1.89v-expressing neurons. No correlation between tau mRNA foci and Venus fluorescence intensity is apparent in hSyn.89v neurons. Same data as in (C). A.U., arbitrary units.

  • Fig. 4 Transient glia cell activation without neurotoxicity upon ZFP-TF.89v tau reduction in vivo.

    (A) mRNA levels of GFAP, Iba1, and Olig1 in hippocampal extracts after ZFP-TF injection. After 6 weeks, GFAP and Iba1 were elevated in all AAV ZFP-TF–injected mice, indicating mild to moderate gliosis. 72v expression generally caused stronger glia activation. After 11 months, compared to 6 weeks, CMV.89v–injected mice showed reduced Iba1 levels. Means ± SEM, one-way ANOVA with Sidak’s test, reference group for significance indicated by arrowhead, n = 3 mice per group. (B) mRNA levels of NeuN, Map1B, and Map2 in hippocampal extracts after AAV ZFP-TF injection. No changes occurred after 6 weeks and 11 months across all groups. mRNA levels are normalized to PBS. Means ± SEM, one-way ANOVA with Sidak’s test, n = 3 mice per group. (C) Time course of ZFP, tau, and cell type–specific mRNA levels after hSyn1.89v compared to PBS injection. A second tau-targeted ZFP-TF, hSyn1.90v, was tested at 4 and 12 weeks p.i. Tau mRNA was stably repressed by ~70% in hSyn1.ZFP-TF–injected mice after 2 weeks. GFAP and Iba1 mRNA increased until 4 weeks and then declined. hSyn1.90v showed substantially less glia transcripts after 12 weeks. NeuN mRNA was unaffected. Lines connect means of hSyn1.89v-, hSyn1.90v-, or PBS-injected hippocampi, and data points represent individual animals, n = 4 mice per time point. (D) Transmission electron microscopy micrographs of axon cross sections in the fornix (white outlined area) of CMV.89v-injected (12 months) and noninjected mouse. No difference in axon diameter was detected, neither across animals [violin plot; median (green) and interquartile range (pink)] nor across groups (bar graph; means ± SEM, two-tailed Student’s t test, n = 3 CMV.89v and n = 2 noninjected mice). Myelination (dark layer around axons) appeared variable across individual axons in each animal.

  • Fig. 5 Rapid brain tau reduction after a single systemic injection of AAV-PHP.B ZFP-TFs.

    (A) Brain sections from mice with a single intravenous (IV) injection of PHP.B hSyn1.GFP show transduction of neurons in various brain regions and spinal cord. Injection of the same dose of AAV-PHP.B CMV.89v also achieves brain-wide expression. (B) Experimental overview of time course of tau reduction using AAV-PHP.B hSyn1.89v and hSyn1.90v. (C) Tau mRNA levels in cortex, hippocampus, and cerebellum of PHP.B hSyn1.89v– and 90v-injected animals from 0 to 24 weeks. PHP.B hSyn1.GFP and PHP.B hSyn1.72v did not reduce tau mRNA. Means ± SEM, two-way ANOVA versus PBS, n = 3 to 8 mice per group and time point, three tissue samples per region and measurement. P value indication: * for 89v; # for 90v. Note that the unexpected low tau mRNA levels in the cerebellum after 4 weeks do not reflect in tau protein levels. (D) Tau protein levels (by ELISA) in cortex, hippocampus, cerebellum, and CSF of PHP.B hSyn1.89v– and 90v-injected animals from 0 to 24 weeks. PHP.B hSyn1.GFP and PHP.B hSyn1.72v did not reduce tau protein. Means ± SEM, two-way ANOVA versus PBS, n = 3 to 8 mice per group and time point, three tissue samples per region and measurement. P value indication: * for 89v; # for 90v.

  • Fig. 6 Sustained tau repression across brain regions without off targets after systemic AAV-PHP.B ZFP-TF injection.

    (A) Tau, ZFP, and cell type–specific (GFAP, Iba1, and NeuN) mRNA levels across brain regions after long-term (6 months) expression of hSyn1.89v, hSyn1.90v, or hSyn1.72v. Mice received one retro-orbital injection of 2.5 × 1012 vg per mouse. Data for tau mRNA are normalized to mice treated with PBS. Data are presented as means ± SEM, two-way ANOVA, n = 6 to 8 mice per group. P value indication: * for 89v; # for 90v. (B) RNA array data from PHP.B hSyn1.89v–, hSyn1.90v-, hSyn1.72v-, and PBS-injected mice compared to PHP.B GFP–injected animals show no significant off-target gene deregulation after 10 weeks (2.5 × 1012 vg per mouse). Threshold for significant down-regulation (pink circles) or up-regulation (green circles) at false discovery rate (FDR)–adjusted P values, P < 0.05. n = 8 to 10 mice per group and n = 19,464 transcript clusters assessed. Raw data are available in table S4.

  • Fig. 7 Global tau reduction by AAV-PHP.B ZFP-TFs protects against Aβ plaque–induced dystrophies.

    (A) Experimental overview: 4-month-old male APP/PS1 mice and age-matched control WT mice were retro-orbitally injected with a low dose (1 × 1012 vg per mouse) of PHP.B hSyn1.89v, PHP.B hSyn1.90v, or PHP.B hSyn1.GFP, and the brain tissue was analyzed for RNA and protein content by immunohistochemistry (IHC) after 8 weeks. (B) Tau and ZFP-TF transcripts in the anterior cortex and hippocampus (HPC) of injected WT and APP/PS1 mice. Means ± SEM, one-way ANOVA, n = 2 to 7 mice per group. (C) Tau protein in cortical and hippocampal lysates of injected WT and APP/PS1 mice analyzed by Western blot. Tau protein is reduced by 20 to 30% in all mice injected with PHP.B hSyn1.89v or 90v. Means ± SEM, two-way ANOVA with Sidak’s test, n = 2 to 7 mice per group. (D) GFP (green) and axonal phospho-neurofilament (SMI312, red)–containing dystrophies around cortical Aβ plaques (blue) in a PHP.B hSyn1.GFP–injected animal. (E) The number of GFP+ dystrophies (immunolabeled with anti-GFP antibodies) per cortical plaque is reduced by ~50% in 89v- and 90v- compared to GFP-expressing APP/PS1 mice. Means ± SEM, two-way ANOVA with Sidak’s test, n = 4 to 7 mice per group, four to five cortical sections per mouse. (F) The number of SMI312+ dystrophies per cortical plaque is similar in 89v- and 90v- compared to GFP-expressing APP/PS1 mice. Means ± SEM, two-way ANOVA with Sidak’s test, n = 4 to 7 mice per group and four to five cortical sections per mouse. (G) Distribution of dystrophy numbers per cortical plaques in all mice and sections [same data as in (E) and (F)]. (H) Colocalization analysis of GFP-filled (green) and SMI312-filled (red) dystrophies around Aβ plaques (blue) in PHP.B hSyn1.GFP–injected APP/PS1 mice reveals that only ~10% of SMI312+ dystrophies originated from neurons transduced with the AAV and expressing GFP. Means ± SEM, n = 6 mice. MW, molecular weight.

  • Fig. 8 Global tau reduction by PHP.B hSyn1.89v or 90v protects against Aβ plaque–induced dystrophies.

    (A) Transcript levels of APP and its processing enzymes BACE1 and PS1 in hippocampal and cortical lysates of WT and APP/PS1 mice injected with AAV-PHP.B GFP, 89v, or 90v. Reduced APP mRNA was detected in the cortex of 89v-expressing APP/PS1 compared to GFP- and 90v-expressing mice. BACE1 and PS1 mRNA was similar across groups. (B) Cortical lysates from APP/PS1 mice with tau reduction contain similar levels of BACE1 protein but reduced APP protein compared to GFP-expressing controls. Aβ:APP ratios are similar across groups. (C) Transcript levels of Aβ peptide–degrading proteases in hippocampal and cortical lysates from AAV-PHP.B GFP–, 89v-, or 90v-expressing mice: mRNA of the endopeptidases neprilysin and Ece1 and the matrix metalloprotease MMP9 are similar across groups. Data in (A) to (C) are presented as means ± SEM, n = 4 to 6 mice, one-way ANOVA with Sidak’s test for multiple comparison.

Supplementary Materials

  • Supplementary Materials

    Persistent repression of tau in the brain using engineered zinc finger protein transcription factors

    Susanne Wegmann, Sarah L. DeVos, Bryan Zeitler, Kimberly Marlen, Rachel E. Bennett, Marta Perez-Rando, Danny MacKenzie, Qi Yu, Caitlin Commins, Riley N. Bannon, Bianca T. Corjuc, Alison Chase, Lisa Diez, Hoang-Oanh B. Nguyen, Sarah Hinkley, Lei Zhang, Alicia Goodwin, Annemarie Ledeboer, Stephen Lam, Irina Ankoudinova, Hung Tran, Nicholas Scarlott, Rainier Amora, Richard Surosky, Jeffrey C. Miller, Ashley B. Robbins, Edward J. Rebar, Fyodor D. Urnov, Michael C. Holmes, Amy M. Pooler, Brigit Riley, H. Steve Zhang, Bradley T. Hyman

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