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

Expression and localization of LB markers are not affected by LRRK2 -/-.

<p>Immunocytochemical localization of Lamp1, ABCa3, and surfactant proteins SP-B and SP-C in ATII isolated from LRRK2 -/- rats (A) and ATII cells isolated from wt rats (B). Lamp1 and ABCa3 localized to LB limiting membrane, whereas SP-B and SP-C are inside the vesicle together with the lipid component of surfactant. No obvious differences in localization of these markers in cells isolated from KO and wt rats were observed. Arrows indicate individual LBs in type II cells. Scale bar = 10 µM</p

DPPC concentration in supernatant as a measure of surfactant secretion.

<p>DPPC concentration was measured in supernatant of unstimulated cells and of cells stimulated with either 100 µM ATP or 300 nM PMA for 30 minutes. DPPC concentration in supernatant was significantly higher in wt cells following stimulation with 100 µM ATP.</p

LBs are significantly enlarged in LRRK2 -/- rats.

<p>(A) LBs (green) in cells that were metabolically labeled with Bodipy phosphatidylcholine (left, asterisks indicate nuclei, scale bar  = 10 µm). LB size was significantly (p<0.006) increased in cells from LRRK2 -/- animals (diameter: 2.26 µm±0.04, n = 4 animals) compared to LBs in cells from wt animals (diameter: 1.95 µm±0.07, n = 4 animals) (middle). Size distribution diagram for LB sizes derived from wt and LRRK2 -/- animals (right, n = 4 animals). (B) LBs (red) in cells that were stained with lipophilic dye Nile red (left, asterisks indicate nuclei, scale bar  = 10 µm). LB size was significantly (p<0.0003) increased in cells from LRRK2 -/- animals (diameter: 2.29 µm±0.03, n = 4 animals) compared to LBs in cells from wt animals (diameter: 1.98 µm±0.03, n = 4 animals) (middle). Size distribution diagram for LB sizes derived from wt and LRRK2 -/- animals (right, n = 4 animals). In all experiments 25 cells were analyzed in each animal corresponding to approx. 350 – 450 LBs per animal. Images were taken from LRRK2 –/- cells.</p

ATP stimulation results in an increased fusion response in LRRK2 -/- cells.

<p>Response is expressed as the fraction of cells with at least 1 fusion within 10(A), and as a number of fusions/cell in responding cells (B). n denotes number of animals. Equal numbers of experiments were conducted in each animal.</p

ATP accelerates LB fusion and increases intracellular Ca²⁺-release in LRRK2 -/- cells.

<p>LB fusion response time histograms (bars) and fura-2 ratios (lines) in response to stimulation of ATII cells. (A – D) Fusion delay histograms from wt and LRRK2 -/- cells following stimulation with 100 µM ATP (A), 300 nM PMA (B), 100 µM ATP and 300 nM PMA (C) and 1 µM ionomycin (D). Stimulation with ATP resulted in a significant left shift in the fusion delay histograms in LRRK2 -/- cells. Delay histograms were derived from pooled data from 3 to 6 animals per experimental condition and from 45 to 256 individual fusions. Cells were stimulated at t = 0 s. Fura-2 traces were derived from 7 to 116 cells. Panels represent wt cells (left), LRRK2 -/- cells (middle) and overlay of wt and -/- delay histograms (right). (E) Resting [Ca²⁺]_c is not significantly enhanced in cells from LRRK2 -/- animals (145.2±27.3 nM, n = 4 in LRRK2 -/- cells and 121.2±26.5 nM, n = 4 in wt cells, respectively, p = 0.55, approx. 15 to 20 cells were analysed for each animal). (F) Changes in [Ca²⁺]_c following stimulation with 100 µM ATP (Cells were stimulated at t = 0 s, n = 3 and 2 for LRRK2 -/- and wt cells respectively). (G) Peak [Ca²⁺]_c following stimulation with 100 µM ATP in LRRK2 -/- cells (n = 3) compared to wt cells (n = 2).</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