An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

BACKGROUND & AIMS: The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson's disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification. METHODS & RESULTS: Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time. CONCLUSION: These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases

Cre independent adult gene expression in DA neurons of TH-tTA and TH-rtTA mice using a AAV-tetO-Venus vector.

<p><b>(A)</b> Scheme of fluorescent DA neuron labelling in non-DOX treated TH-tTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(B)</b> Confocal fluorescent pictures of Venus expression (green) in DA neurons co-stained for tyrosine hydroxylase (TH; red) in the substantia nigra pars compacta (SNpc) of sagittal TH-tTA mouse brain sections. <b>(C)</b> Scheme of fluorescent DA neuron labeling in DOX-treated TH-rtTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(C)</b> Scheme of fluorescent DA neuron labelling in DOX-treated TH-rtTA mice stereotactically injected with AAV-tetO-Venus vector in the ventral midbrain. <b>(D)</b> Confocal fluorescent pictures of Venus expression in DA neurons co-stained for TH in the substantia nigra pars compacta (SNpc) of sagittal TH-rtTA mouse brain sections. <b>(E)</b> Quantification reveals that in the ON-state 67% and 73% and in the OFF-state 27% and 23% of TH+ cells are also Venus-positive in TH-tTA and TH-rtTA mice, respectively. In non-transgenic mice (controls) only 5% of TH+ cells were also Venus-positive. n = 3–5. Scale bars: 250 μm and in high magnification picture 50 μm.</p

Efficient fluorescent labeling of DA neurons and their axons in AAV-LSL-mCherry injected DA neuron-specific Cre mice.

<p><b>(A)</b> Scheme for specific fluorescent labeling of DA neuron in mice expressing a DA neuron-specific Cre and stereotaxic injected in the mindbrain with a recombinant AAV vector expressing mCherry after a floxed STOP codon. <b>(B-G)</b> Fluorescent mCherry expression in DA cells of the midbrain (B-D) and DA fibers in the striatum (E-G) in sagittal sections of stereotaxic injected DAT-Cre mice. Expression was evaluated 1 (B,E), 3 (C,F) and 5 weeks (D,G) after injection. After 1 week, no expression could be detected in DA fibers of the striatum. Expression in DA fibers was saturated 5 weeks after injection. Scale bars = 200 μm (B-D); 20 μm (E-G). <b>(H-O)</b> Fluorescent mCherry expression in DA cells of the midbrain (H-K) and DA fibers in the striatum (L-O) after stereotaxic injection of TH-tTA/LC1 mice. Fluorescent expression was evaluated at the indicated time points in mice raised without DOX. Some mice were kept on DOX-containing food after the virus injection until analyzed (J, K, N, O). Scale bars = 200 μm (H-K); 20 μm (L-O). <b>(P and Q)</b> No fluorescent mCherry expression was detected in the lateral habenula (LHb) of AAV-LSL-mCherry injected DAT-Cre (P) and TH-tTA/LC1 (Q) mice after 5 weeks. Scale bars = 200 μm.</p

Specificity, inducibility and efficacy of gene expression in TH-tTA/LC1/Rosa26R and TH-rtTA/LC1/Rosa26R mice.

<p><b>(A)</b> Genetic scheme of DOX-regulated reporter gene expression in TH-tTA and TH-rtTA mice crossed with LC1 and Rosa26R reporter mice. <b>(B and C)</b> Coronal midbrain brain sections with substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) DA neurons of TH-tTA/LC1/Rosa26R (B) and TH-rtTA/LC1/Rosa26R (C) mice raised with or without doxycycline (DOX) or transiently with DOX to have the system switched off during development (TH-tTA mice treated with DOX during pre- and postnatal development till 6 weeks of age; TH-rtTA mice raised without DOX) and on during analysis (TH-tTA mice from the age of 6 weeks without DOX; TH-rtTA mice from the age of 6 weeks with DOX). Sections were stained for β-galactosidase activity with X-gal to visualize cells with activated tet-system. Adjacent sections were X-gal stained and co-stained with tyrosine hydroxylase (TH) antibodies and DAB substrate (brown) to mark DA neurons. <b>(D)</b> Zoom in picture of TH and lacZ double positive cells in the SNpc. <b>(E and F)</b> Quantification of TH and lacZ double positive cells (LacZ+TH+) in the SNpc and VTA to estimate recombination efficacy in DA neurons (TH+) of animals treated constantly (constant DOX), transiently (transient DOX) or without DOX (no DOX) in the tet-OFF (E) and tet-ON mice (F). mean + s.e.m.; n = 3. Scale bars: 500 μm (B and C), 50 μm (D).</p

Schematic overview of utilized transgenic mice and recombinant AAV (rAAV) constructs.

<p>TH-tTA and TH-rtTA mice express, under the tyrosine hydroxylase (TH) promoter, the tetracycline-regulated transactivator protein (tTA) or the reverse tetracycline-regulated transactivator protein (rtTA) in dopaminergic (DA) neurons. tTA binds to the tetracycline operon (tetO) in the absence of doxycycline (DOX) and rtTA binds in the presence of DOX, driving transient expression of gene X, for example the fluorescent protein Venus from a recombinant AAV vector (AAV-tetO-Venus), or luciferase and Cre from the LC1 construct. The LC1 encodes a luciferase enzyme, which can be used for bioluminescence <i>in vivo</i> imaging, and the Cre recombinase for deleting gene Y or removing floxed STOP codons (LSL). Cre is used here as a genetic switch to activate, from the ROSA locus, either expression of the reporter gene lacZ (encoding β-galactosidase) to visualize transgene expression or to activate expression of diphtheria toxin protein A (dt-a) to selectively induce cell death of DA neurons. Here we use Cre also to activate expression of the fluorescent protein mCherry from AAV-LSL-mCherry.</p

Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis

Image texture parameter “granularity” for analysis of early mHTT aggregation.

<p>(<b>A</b>) Image analysis strategy for early aggregation parameter “granularity”. The area of cell nuclei was detected based on the DAPI signal. The spatial pattern of pixel intensities of nuclear EM48-ir signal was analyzed and a granularity index indicating formation of small EM48-ir inclusion species was calculated using the SER (Spots) texture algorithm (Acapella, PerkinElmer). Brain sections from 2–12 months old zQ175 heterozygote mice were immunolabelled for DARPP-32 and EM48. The “granularity” index in the striatum and cortex was used to detect and monitor early changes in mHTT distribution and signal clustering. (<b>B</b>) A significant increase in the nuclear EM48 “granularity” in the striatum was observed from 2 to 6 months of age followed by a significant decrease from 6 to 12 months. (<b>C</b>) In the cortex, a significant increase in EM48 granularity was observed from 6 to 12 months. Data are displayed as dot plots with mean +/-SD. Statistical analysis performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every age an n of 8 animals with 6 sections per animal were used for quantitation; *p<0.05; **p<0.01; ***p<0.001.</p

Time course of mHTT inclusions appearance in the striatum and cortex of zQ175 mice.

<p>Brain samples from 3–12 months old zQ175 heterozygous mice were stained for mHTT inclusions (bright spots) by EM48-ir and imaged on the Opera high content microscope. A progressive increase in EM48 signal in both striatal and cortical brain regions was clearly visible with age. mHTT inclusions as indicated by EM48-ir puncta appeared earlier and with higher abundance in the striatum as compared to cortex.</p

Modulation of HTT levels, as evidenced by MAB2174 and EM48 immunoreactivity in the striatum of zQ175 heterozygous mice by AAV2 viruses expressing HTT-targeting shRNAs.

<p>AAV2 viruses encoding GFP and shRNAs directed against HTT were injected in the right ventricle of neonate zQ175 heterozygous mice. At 4 months of age, mice were euthanized and analysed for HTT inclusions and HTT cytoplasmic levels. Representative images showing DARPP-32, mHTT IHC staining (<b>A</b>) or DARPP-32, HTT (MAB2174) IHC staining (<b>B</b>) in the GFP positive striatal region transduced with AAV2 encoding shRNA against HTT (mHtt-sh#2 and mHtt-sh#4) or non-target control shRNAs (shC004). Quantitative analysis of mHTT inclusions (<b>C</b>) and MAB2174-ir intensity (<b>D</b>) in GFP positive cells of the striatum: mHtt targeting shRNA led to a significant decrease in the number of nuclear HTT EM48-ir inclusions and MAB2174-ir HTT cytoplasmic levels in comparison to the control AAV/shC004. Data are displayed as bar graphs with mean +/-SD. Statistical analysis was performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every group an n of 4 animals with 3 sections per animal were used for quantitation; p<0.05; **p<0.01; ***p<0.001.</p

Illustration of inclusion quantification in the striatum of zQ175 mice.

<p>Micrographs showing image segmentation strategy for the automated analysis of mHTT inclusion numbers, localization and size in the striatum. (<b>A</b>) Coronal brain sections were co-immunostained for detection of DAPRPP-32 and mHTT and image acquisition of striatal and cortical regions was performed with the Opera (PerkinElmer Inc.). (<b>B</b>) Multi-field images were acquired using 40x objective lens corresponding to the region of interest for subcellular resolution. (<b>C</b>) Area of cell nuclei was determined based on the DAPI signal using a sliding parabola filter for background correction. (<b>D</b>) Medium spiny neurons were identified using nuclear intensity of DARPP-32 staining (green). (<b>E</b>) An extranuclear region was defined with 10 pixels spacing to the nuclear region. Numbers of mHTT nuclear and extranuclear inclusions in medium spiny neurons (MSNs) were quantified based on EM48 signal.</p