Biochemical analyses of soluble and pellet fraction protein extracts from spinal cord, brainstem and anterior brain reveal no difference in α-synuclein species.

<p>A. Immunoblot of mouse spinal cord lysates detecting soluble α-synuclein monomers, oligomers/aggregates, truncations and S129-phosphorylation (P-aSN). B. Immunoblot of mouse spinal cord lysates detecting soluble HSP70. C. Immunoblot of soluble (supernatant) and pellet fractions of brainstem detecting HSP70 and α-synuclein monomers, oligomers/aggregates, truncations and S129-phosphorylation. D. Immunoblot of soluble (supernatant) and pellet fractions of anterior brain detecting HSP70 and α-synuclein monomers, truncations and S129-phosphorylation (P-aSN). β-actin was used as loading control. The size markers are indicated. E. Bar graphs representing quantitative analyses of the immunoblots shown in C and D for the α-synuclein monomers, oligomers/aggregates, truncated forms and S129-phosphorylation (P-aSN) normalized to the loading control β-actin in soluble and pellet fractions. n = 5 per genotype. Asteriks indicate statistical significance (*: p<0.05); student's t-test, two-tailed. Error bars: standard deviation.</p

Histological analyses reveal no difference in α-synuclein distribution/localization in the brain.

<p>Brain sections of single and double transgenic Thy1-HSP70/Thy1-haSN(A53T) mice stained against human α-synuclein. Black arrow: cytoplasmic and nuclear α-synuclein in the CA1 area of the hippocampus and brainstem neurons. Scale bar: 100 µm.</p

HSP70 overexpression in an α-synucleinopathy mouse model is not beneficial but even worsens the phenotype.

<p>Measurement of weight, forelimb grip strength and motor coordination (rotating beam and rotarod) of single- and double-transgenic Thy1-HSP70/Thy1-haSN(A53T) mice. Asteriks indicate statistical significance (*: p<0.05, **: p<0.01; student's t-test, two-tailed). Error bars: SEM.</p

Thy1-HSP70 mice appear and behave normally and are phenotypical indifferent to wildtype mice.

<p>Measurement of weight, forelimb grip strength and motor coordination (rotating beam and rotarod) of single Thy1-HSP70 and wildtype mice. Error bars: SEM.</p

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

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