Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

<div><p>Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.</p></div

mHtt gene-dose impacts on DARPP32 protein levels.

<p>DARPP32 immunostaining in sagittal mouse brain sections of 8-month-old wildtype (A), HdhQ150 HET (B), and HdhQ150 HOM mice (C). Staining was performed by automated paraffin immunohistochemistry using the Ventana Discovery XT technology and DAB as chromogen. Overall DARPP32 staining intensities clearly decline from wildtype (A) to HdhQ150 HET (B) and HOM mice (C), and in all DARPP32⁺ brain regions including striatum (Str), cortex (Ctx), thalamus (Tha), and cerebellum (Cer). The images are representative of results obtained from 2 WT, 6 HdhQ150 HET and 6 HdhQ150 HOM mice. (D) Representative western blot of DARPP32 protein levels in 6-month-old wildtype (WT), HdhQ150 HET and HOM mice, 3 of each. (E) Quantification of Western blot signals (normalized to wildtype and loading control β-actin) revealed a highly significant reduction of DARPP32 levels in HdhQ150 striatum, as compared to wildtype striatum (n = 6, p<0.005). The difference between HdhQ150 HET (n = 9) and HdhQ150 HOM (n = 6) DARPP32 levels was also statistically significant (p<0.01). Quantification results are the average of three independent Western blots. A second control (tubulin) shows that DARPP32 changes are not due to differences in loading. Statistics: One-way ANOVA, Holm-Sidak's multiple comparisons test</p

MW8⁺ mHtt aggregates in brain regions of HdhQ150 mice.

<p>MW8 immunofluorescence (in red) in frozen sections of 8-month-old HdhQ150 HET (A, C, E, G, I) and HdhQ150 HOM mice (B, D, F, H, K). Numerous neuronal intra-nuclear inclusions (NIIs) are visible in striatum (A, B), olfactory bulb (C, D), the CA3 region of the hippocampus (E, F) and in cerebellum (G, H). NIIs are much larger in HOM (B, D, F, H) as compared to HET mice (A, C, E, G). The dentate gyrus of HdhQ150 HOM mice (K) shows numerous extra-nuclear mHtt aggregates in the polymorph layer (PoDG) and large inclusions in the granular layer (GrDG). Such deposits are rare in the dentate gyrus of age-matched HdhQ150 HET mice (I). The large, elongated and irregularly shaped structures are blood vessels (e.g. see arrow in insert of I). These are non-specifically stained due to cross-reactivity of the secondary antibody with mouse IgG. The images are representative of results obtained from 6 HdhQ150 HOM and 6 HdhQ150 HET mice. Sections were counter-stained with DAPI (blue).</p

GFAP⁺ astroglial cells in the hind brain of HdhQ150 mice.

<p>Images show the results of GFAP immunohistochemistry on sagittal brain sections of 8-month-old wildtype (A, B), HdhQ150 HET (C, D), and HdhQ150 HOM mice (E, F). Staining was performed by automated immunohistochemistry using the Ventana Discovery XT technology and DAB as chromogen. As compared to wildtype (A, B), GFAP staining is dramatically increased in the brainstem (BS) of HdhQ150 HET mice (C, D) and the increase is much more pronounced in HdhQ150 HOM mice (E, F). Note that the striatum (Str) in HdhQ150 mice is largely devoid of activated astroglia. The images are representative of results obtained from 2 wildtype, 6 HdhQ150 HOM and 6 HdhQ150 HET mice.</p

mHtt aggregates in DARPP32⁺ striatal neurons of HdhQ150 and R6/2 mice.

<p>Images represent paraffin sections of striatum, double stained (immunofluorescence) with anti-DARPP32 (green) and MW8 to visualize neuronal intra-nuclear inclusions (NIIs; shown in red). Shown are central regions of the striatum of a 10-month-old wildtype mouse (WT; A), a 10-month-old HdhQ150 HET mouse (B) and a 12-week-old R6/2 mouse (C). NIIs are visible in most MSNs but absent in neurons with high DARPP32 staining signals (arrows). The images are representative of results obtained from 3 WT, 3 HdhQ150 HET and 3 R6/2 mice.</p

mHtt aggregates in the HdhQ150 mouse retina.

<p>Images show MW8 immunofluorescence in frozen sections of the retina of a wildtype mouse (A), a 6-month-old HdhQ150 HOM mouse (B), and a 10-month-old HdhQ150 HET mouse (C). Aggregates (green dots) are visible only in the HdhQ150 mouse retinas where these are present in all neuronal cell layers and cell types including photoreceptors, inner nuclear and ganglion cells. No aggregates were detected in non-neuronal cell layers such as choroid or pigment epithelium. Aggregate size and number is similar in 6-month-old HdhQ150 HOM and 10-month-old HdhQ150 HET mouse retinas. (D) High magnification image of C showing that retinal neuron mHtt deposits are mainly intra-nuclear (NIIs). MW8 staining results at higher magnification show mHtt inclusions (green dots) on a blue background of DAPI-stained nuclei. Large irregularly shaped green areas are due to non-specific cross-reactivity of the secondary antibody with mouse IgG. Images are representative of results obtained from 4 HdhQ150 HET and 3 HdhQ150 HOM mice.</p

Iba 1⁺ microglial cells in the hind brain of HdhQ150 mice.

<p>Images represent Iba1 immunohistochemical staining results in sagittal brain sections of 8-month-old wildtype (A, B), HdhQ150 HET (C, D), and HdhQ150 HOM mice (E, F). Staining was performed by automated immunohistochemistry using the Ventana Discovery XT technology and DAB as chromogen. As compared to wildtype (A, B), Iba 1 staining is more pronounced in brainstem and cerebellar nuclei of HdhQ150 mice and markedly increased in HdhQ150 HOM as compared to HdhQ150 HET mice. The brainstem and cerebellar nuclei of HdhQ150 mice contain numerous large Iba1⁺ cells with an ameboid morphology reminiscent of activated microglia. The images are representative of results obtained from 2 wild type, 6 HdhQ150 HOM and 6 HdhQ150 HET mice.</p

High LRRK2 transgene levels do not exacerbate α-synuclein-driven phenotypes.

<p>(A) Schematic representation of the four different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated measures ANOVA (group effects for the respective panels: 1: F(1,22) = 0.483, p = 0.494; 2: F(1,26) = 0.000, p = 0.983; 3: F(1,11) = 0.738, p = 0.409; 4: F(1,22) = 2.048, p = 0.166; 5: F(1,16) = 1.255, p = 0.279; 6: F(1,27) = 5.171, p = 0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice had to be killed because of too severe motor deficits (1 = 100% and 0 = 0% of mice alive). The different genotypes, gender, number of mice per genotype and the p-values (nonparametric Kaplan-Meier) are indicated.</p

aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic mice.

<p>Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total α-synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). β-actin (βAc) levels were measured as loading control and for normalization. For reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different α-synuclein protein species/forms are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive proteins (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone.0036581.s007" target="_blank">Figure S7</a>). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to βAc. Circles represent individual mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by two-tailed, unequal variances Student’s t-test. Genotypes: aSN = haSN(A53T), aSN/LRRK2 = haSN(A53T)/hLRRK2(G2019S), Ntg = non-transgenic wildtype littermate control and KO = aSN knock-out mice.</p

Motor assessment and aSN/Tau protein characterization in hLRRK2(G2019S) mice.

<p>(A) Motor skill learning of 4-month-old male and 6-month-old female hLRRK2(G2019S) and Ntg controls in the 3-step accelerated rotarod task over four consecutive days. The number of mice per genotype is indicated. Three batches of animals were included in this graph (single transgenic and Ntg animals from experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone-0036581-g003" target="_blank">Figure 3B</a> as well as a separate batch). p-values were determined by repeated measure ANOVA (group effect males: F(1,119) = 9.42, p = 0.003, group effect females: F(1,52) = 3.74, p = 0.059). (B) Novelty-induced horizontal locomotor activity of 7.3- and 28.2-month-old hLRRK2(G2019S) and Ntg mice. Bar graphs show the sum of the distance travelled from 5–30 min and from 35–60 min. The number of mice per genotype is indicated. p-values were determined either by repeated measure ANOVA (group effect males 7.3 M: F(1,16) = 4.044, p = 0.061; group effect males 28.2 M: F(1,16) = 0.093, p = 0.764) or by two-tailed, unequal variances Student’s t-test. (C) Western blotting of forebrain homogenates from 15-month-old hLRRK2(G2019S) (TG) and Ntg male mice. Lower panel: Shown are levels of mouse α-synuclein (aSN) and phospho-α-synuclein Ser129 (paSN) as well as mouse microtubule-associated protein Tau and phospho-Tau Ser202/Thr205 (pTau). β-actin (βAc) was used as loading control and for normalization. Upper panel shows the results of the immunoblot quantifications. Circles represent individual mice, the means (% normalized to Ntg) are indicated as horizontal bars. p-values were determined by two-tailed, unequal variances Student’s t-test. Ntg: non-transgenic wildtype littermate control.</p