Genetic manipulation restores MT stability and rescues fibroblast phenotype.

<p>(A) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with parkin mutations (PARK) transfected with control plasmid (VEC) or WT parkin (WT), and of control fibroblasts (CONT) transfected with short hairpin RNA, sh-183 (183) or control shRNA (VEC). (B-D) Morphometric analyses of patients fibroblasts expressing control plasmid (PARK-VEC, N = 4) or WT parkin (PARK-WT, N = 4), and control fibroblasts transfected with control shRNA (CONT-VEC, N = 4) or silenced with sh-183 (CONT-183, N = 4), showing the ratio between maximum and minimum axes (B), the area (C) and the number of overlapping regions between cells (D). ns = not significant, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. (E) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with LRRK2 mutations (LRRK2) transfected with control plasmid (VEC) or WT LRRK2 (WT), and of control fibroblasts (CONT) expressing control plasmid (VEC) or G2019S mutant LRRK2 (MUT). Morphometric analyses of patients fibroblasts expressing control plasmid (LRRK2-VEC, N = 3) or WT LRRK2 (LRRK2-WT, N = 3), or of control fibroblasts transfected with control plasmid (CONT-VEC, N = 3) or G2019S mutant LRRK2 (CONT-MUT, N = 3), showing the ratio between maximum and minimum axes (F), the area (G) and the number of overlapping regions between cells (H). ns = not significant, *<i>p</i><0.05, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM.</p

PD fibroblasts show subtle cytoskeleton differences.

<p>(A) Immunoblot and (B) densitometric analyses of vimentin (Vim), α-tubulin (α-Tub), β-tubulin (β-Tub) and actin (Actin) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 10), mutated <i>parkin</i> (PARK, dark grey bars, N = 6), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 6) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of each protein were normalized on the level of GAPDH of the relative sample. All values are expressed as mean ± SEM. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control, ##<i>p</i><0.02 vs PD, according to ANOVA, Tukey HSD <i>post hoc</i> test. (C) Cultured human fibroblasts were stained with anti-vimentin and anti-α-tubulin primary antibodies or with TRITC-conjugated phalloidin to reveal the organization of intermediate filaments (Vim, top), microtubules (α-Tub, middle) and actin fibers (Actin, bottom), respectively. Concurrent nuclear staining was made by using DAPI (Blue). Scale bar: 20 µm.</p

GSK3β phosphorylation is reduced in PD fibroblasts.

<p>(A) Immunoblot and densitometric analyses of (B) total and (C) phosphorylated glycogen synthase kinase 3 beta (GSK3β), p38 MAP Kinase (p38) and p44/42 MAPK (Erk) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 3), mutated <i>parkin</i> (PARK, dark grey bars, N = 3), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 3) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of total protein were normalized on the level of GAPDH of the relative sample, whereas the levels of phosphorylated form were normalized on the values of total protein. All values are expressed as mean ± SEM. *<i>p</i><0.05 and **<i>p</i><0.02 vs control, #<i>p</i><0.05 vs PD according to ANOVA, Tukey HSD <i>post hoc</i> test.</p

Morphological alterations characterize PD fibroblasts.

<p>(A) Representative phase contrast micrographs of cultured human fibroblasts of healthy and PD affected people. Scale bar: 25 µm. Morphometric analysis showed reduced ratio between maximum and minimum axes in parkinsonian fibroblast (B) and increased area in the presence of mutated parkin or LRRK2 (C). (D) Histogram showing the increased number of overlapping regions between cells in patient fibroblasts. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. CONT = control (N = 10); PARK = patients with mutations of <i>parkin</i> (N = 6); LRRK2 = patients carrying mutations in <i>LRRK2</i> (N = 6); PD = idiopathic Parkinson’s disease patients (N = 3).</p

Correlation between phase shift modulation at different gait velocities and DAT binding values of the putamen (A) and caudate nucleus (B).

<p>Full dots represent arms with reduced (<18°) ROM when walking at preferred gait speed. Empty dots shows arms with arm ROM within the normal range. When arm ROM was reduced, a positive correlation was found between upper-lower limb phase shift modulation and both DAT binding values of putamen (RSquare = 0.37, p = 0.01) and caudate nucleus (RSquare = 0.38, p = 0.01). Correlation lines for arms with normal ROM (>18°) are not shown.</p

ASA significantly correlated to the AI of DAT binding values of the putamen (see, Methods).

<p>ASA significantly correlated to the AI of DAT binding values of the putamen (see, Methods).</p

Spatio-temporal parameters of the stride, arm ROM and phase shift of subjects with Parkinson disease and healthy subjects.

<p>Median values, non-outlier min-max, and levels of statistical difference (Mann-Whitney U-test or Matched pairs) are reported. Data refer at walking at different velocities unless otherwise specified.</p>*<p>p<0.05 (PD vs. HC); ** p<0.01 (PD vs. HC). BH = body height (mm). ROM = range of motion; Phase shift = temporal delay (%stride) between the positive peak (antero-posterior swing) of the wrist and the negative peak of malleolus; Stride = the period from initial contact of one foot and following initial contact of the same foot, is one gait cycle. Stance = gait phase when a foot is in contact with the ground, it begins with initial heel contact and ends with toe off.</p><p>For Parkinson patients, ipsilateral and contralateral refers to the more dopamine depleted hemisphere. For healthy controls, left hemibody refers conventionally to ipsilateral.</p

CONSORT flow diagram.

<p>CONSORT flow diagram.</p

Rehabilitation in progressive supranuclear palsy: Effectiveness of two multidisciplinary treatments

<div><p>Background</p><p>to date, there are no medical or surgical treatments for progressive supranuclear palsy (PSP). It is possible to speculate that patients with PSP could benefit from rehabilitative treatments designed for Parkinson’s disease, including the use of robot-assisted walking training.</p><p>Objective</p><p>to evaluate whether the use of the robotic device Lokomat® is superior in PSP patients to the use of treadmill with visual cues and auditory feedbacks (treadmill-plus) in the context of an aerobic, multidisciplinary, intensive, motor-cognitive and goal-based rehabilitation treatment (MIRT) conceived for Parkinsonian patients.</p><p>Methods</p><p>we enrolled twenty-four PSP patients. Twelve subjects underwent a 4-week MIRT exploiting the use of the treadmill-plus (MIRT group). Twelve subjects underwent the same treatment, but replacing the treadmill-plus with Lokomat® (MIRT-Lokomat group). Subjects were evaluated with clinical and functional scales at admission and discharge. The primary outcomes were the total PSP Rating Scale (PSPRS) score and its “limb” and “gait” sub-scores. Secondary outcomes were Berg Balance Scale (BBS), Six Minutes Walking test (6MWT) and the number of falls.</p><p>Results</p><p>total PSPRS, PSPRS-gait sub-score, BBS, 6MWT and number of falls improved significantly in both groups (p ≤ 0.003 all, except 6MWT, p = 0.032 and p = 0.018 in MIRT-Lokomat and MIRT group respectively). The PSPRS-limb sub-score improved significantly only in the MIRT group (p = 0.002). A significant difference between groups was observed only for total PSPRS, indicating a slightly better improvement for patients in the MIRT group (p = 0.047). No differences between groups were revealed for the other outcomes, indicating that the effect of rehabilitation was similar in both groups.</p><p>Conclusions</p><p>Lokomat<b>®</b> training, in comparison with treadmill-plus training, does not provide further benefits in PSP patients undergoing MIRT. Our findings suggest the usefulness of an aerobic, multidisciplinary, intensive, motor-cognitive and goal-based approach for the rehabilitation of patients suffering from such a complex disease as PSP.</p><p>Trial Registration</p><p>This trial was registered on ClinicalTrials.gov, <a href="https://clinicaltrials.gov/ct2/show/NCT02109393" target="_blank">NCT02109393</a>.</p></div

Baseline and end of treatment values of outcome variables for both groups of patients.

<p>Reported p-values are computed by the Wilcoxon signed rank test. Data are reported as median (lower quartile, upper quartile).</p