Characterization of the samples included in this study.

<p>Characterization of the samples included in this study.</p

Trehalose induces autophagy and activates autophagic flux in CHIP-mutant fibroblasts.

<p>The cells were treated for 24 h with o without trehalose 50 mM in the presence or absence of 10 µM chloroquine (CQ), a lysosomotropic agent widely used to block lysosomal degradation. <b>A</b>) Fluorescence microscopy images showing autophagosome marker LC-3 (green), lysosomal marker Lamp-2A (red) and LC3-LAMP-2A colocalization (yellow). (Scale bar  = 20 µm). <b>B</b>) Integrated optical density (IOD) quantification of LC3 expression. <b>C</b>) The lysosomal degradation was measured by IOD quantification of colocalization LC-3-LAMP-2A (yellow). <b>D</b>) Western blot showing LC3-II accumulation. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. In 7B and 7C values are the mean ± S.E.M. of six independent coverslips (each value represents the mean of 20 field for coverslip). Three individuals with the mean of 2 independent coverslips (n = 3) in the control group and in the CHIP-mutant group 6 independent coverslips of the only patient (pseudo-replicates, n = 6). In 7D values represents the mean of 2 independent dishes of cells of 2 controls (n = 2) and for the CHIP-mutant group one patient with four independent dishes (pseudo-replicates, n = 4). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents without chloroquine; δδp<0.01, δδδp<0.001 trehalose -CQ <i>vs</i> trehalose + CQ treated cultures.</p

ROS production, Glutathione homeostasis and Mitochondrial membrane potential.

<p><b>A</b>) Representative images of DCF staining in control and CHIP-mutant fibroblasts for the indicated treatment. Scale bar  = 10 µm. <b>B</b>) Percentage of DCF positive fibroblasts. One hundred fifty to two hundred cells per coverslip cells were counted. <b>C</b>) GSH levels in control and CHIP mutant human fibroblasts with 7 and 12 passes. <b>D</b>) Representative images of MitoTracker TM Ros and nuclei Hoescht staining in control and CHIP-mutant fibroblasts cultured in the presence of epoxomicin (10 nM) and/or trehalose (50 mM). Yellow arrows indicate small depolarized mitochondria (scale bar  = 30 µm). <b>E</b>) Quantification in IOD of fibroblasts with altered mitochondria morphology by fluorescence imaging analysis in 10 random fields of 6 coverslips from control and CHIP-mutant cultures (Scale bar  = 15 µm). <b>F</b>) Complex IV staining showed an abundant and granular perinuclear pattern indicating mitochondrial compartmentalization (scale bar  = 10 <i>µ</i>m). A cell of this micrograph has been magnified in the pictures below. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values of 3B are the mean of two independent experiments of 4 independent cell dishes “n = 4” (pseudo-replicates) of the patient group and in control group “n = 2”: the mean of 2 different controls with 4 dishes of cells each one. In 3C and 3E values are the mean ± SEM from two experiments with 6 independent cells dishes (n = 6, pseudo-replicates). Statistical analysis was performed by Student's t-test. *p<0.01, **p<0.01, ***p<0.001 vs solvents; δp<0.05, δδp<0.01, δδδp<0.001 trehalose + epoxomicin vs epoxomicin treated cultures; ∧∧∧p<0.001 fibroblasts with 12 <i>vs</i> 7 passages.</p

Trehalose increases autophagy activity in CHIP-mutant human fibroblasts.

<p>After 5 DIV, fibroblasts were pre-treated with trehalose (50 or 100 mM) for 15 minutes followed by addition of epoxomicin or solvent for another 24 h. <b>A</b>) LC3 staining (green) and nuclei (Hoescht, blue) immunofluorescence in CHIP-mutant fibroblast treated with trehalose (100 mM) and their quantification <b>B</b>) in percentage of cells with more than 50 autophagic vesicles (Scale bar  = 20 µm). <b>C</b>) Representative microphotographs of Lysosomal-associated membrane protein, LAMP-2A, present in lysosomes and endosomes, (Scale bar  = 20 µm) and <b>D</b>) quantification in percentage of cells with LAMP-2A positive vesicle distribution in the perinuclear region, around the nucleus. <b>E</b>) Images of fibroblasts expressing CD63, as a late endo/lysosomal marker (Scale bar  = 30 µm) and <b>F</b>) quantification in percentage of optical intensity for CD63 inmunoreactivity. The insets show boxed regions at higher magnification (Scale bar  = 15 µm). <b>G</b>) Immunofluorescence for HSC70 and LAMP-2A in CHIP-mutant cultures treated with trehalose 100 mM for 48 h (Scale bar  = 20 µm) and <b>H</b>) percentage of co-localization LAMP-2A/HSC70 positive cells. <b>I</b>) Percentage of LAMP-2A positive cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values in 5B, 5D, 5H and 5I are the mean ± S.E.M. of two experiments with six cover slips, from 150 to 250 cells of every group were counted. In 5F, in CD63 immunostaining data are the mean of 4 independent dishes of cells for 2 control individuals (n = 2), in the CHIP-mutant group 6 independent dishes of cells of the only patient (pseudo-replicates, n = 6). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents; δp<0.05, δδδp<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin treated cultures.</p

Proliferation and apoptosis of human fibroblasts with compound heterozygous CHIP mutations.

<p>Trehalose protects from apoptosis and epoxomicin-induced cell death in CHIP-mutant human fibroblasts. <b>A</b>) Photomicrographs of dose-dependent effects of epoxomicin and trehalose pre-treatment 15 min before epoxomicin for 24 h on the expression of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts (scale bar  = 30 µm). <b>B</b>) Percentage of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts treated with 0, 2 and 10 nM epoxomicin for 24 h and trehalose 50 mM pre-treatment after 5 days in vitro. One hundred fifty to two hundred cells per coverslip cells were counted. <b>C</b>) Photomicrographs of dividing BrdU⁺ cells and total nuclei stained with bis-benzimide. (Scale bar  = 30 µm). <b>D</b>) Percentage of BrdU⁺ cells with respect to the total number of fibroblasts with 7 and 12 passages, respectively. Sixty to eighty cells were counted per coverslip to obtain the percentages of BrdU⁺ cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values are the mean of two experiments of 6 independent cell dishes (pseudo-replicates, n = 6) of the patient group and in control group: the mean of the 3 different controls with 3 dishes of cells each one (n = 3). Statistical analysis was performed by Student's t-test. **p<0.01, ***p<0.001 <i>vs</i> solvents; δδδ p<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin; ∧∧∧p<0.001 fibroblasts with 12 <i>vs</i> 7 passages.</p

Expression of p62, Atg5–Atg12 and Beclin-1 in controls and CHIP-mutant fibroblasts.

<p>Trehalose treatment increases the autophagic activity. Fibroblasts were exposed to 50 mM trehalose for 24 hours and the autophagy-related proteins were analyzed by Western blot. <b>A</b>) Western blot showing the autophagic degradation of p62, a major selective substrate for autophagy. <b>B</b>) Beclin-1 and <b>C</b>) Atg5-12, essential factors for autophagosome formation were increased by trehalose. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean ± SEM. Values are the mean of two experiments of independent dishes of one patient (pseudo-replicates, n = 4–6) and for control group 3 different controls with the mean of different dishes of cells (n = 3). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents; δδp<0.01, δδδp<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin treated cultures.</p

Additional file 1: FigureS1. of Genotype-phenotype correlations and expansion of the molecular spectrum of AP4M1-related hereditary spastic paraplegia

Expression of the AP4M1 gene in several regions of the human brain throughout development and aging. Note the higher expression levels during fetal development (birth is marked with a vertical solid line). Data from the Human Brain Transcriptome (HBT) project ( http://hbatlas.org ). CBC - cerebellar cortex, MD - mediodorsal nucleus of the thalamus, STR - striatum, AMY - amygdala, HIP - hippocampus, and NCX – neocortex. (PDF 68 kb