Early detection of structural abnormalities and cytoplasmic accumulation of TDP-43 in tissue-engineered skins derived from ALS patients.
Bottom Line: As a result, the identification and development of disease-modifying therapies is difficult.Aiming to generate an innovative human-based model to facilitate the identification of predictive biomarkers associated with the disease, we developed a unique ALS tissue-engineered skin model (ALS-TES) derived from patient's own cells.Remarkably, these abnormal skin defects, uniquely seen in the ALS-derived skins, were detected in pre-symtomatic C9orf72-linked ALS patients carrying the GGGGCC DNA repeat expansion.
Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the selective degeneration of motor neurons in the brain and spinal cord progressively leading to paralysis and death. Current diagnosis of ALS is based on clinical assessment of related symptoms. The clinical manifestations observed in ALS appear relatively late in the disease course after degeneration of a significant number of motor neurons. As a result, the identification and development of disease-modifying therapies is difficult. Therefore, novel strategies for early diagnosis of neurodegeneration, to monitor disease progression and to assess response to existing and future treatments are urgently needed. Factually, many neurological disorders, including ALS, are accompanied by skin changes that often precede the onset of neurological symptoms. Aiming to generate an innovative human-based model to facilitate the identification of predictive biomarkers associated with the disease, we developed a unique ALS tissue-engineered skin model (ALS-TES) derived from patient's own cells. The ALS-TES presents a number of striking features including altered epidermal differentiation, abnormal dermo-epidermal junction, delamination, keratinocyte infiltration, collagen disorganization and cytoplasmic TDP-43 inclusions. Remarkably, these abnormal skin defects, uniquely seen in the ALS-derived skins, were detected in pre-symtomatic C9orf72-linked ALS patients carrying the GGGGCC DNA repeat expansion. Consequently, our ALS skin model could represent a renewable source of human tissue, quickly and easily accessible to better understand the physiophatological mechanisms underlying this disease, to facilitate the identification of disease-specific biomarkers, and to develop innovative tools for early diagnosis and disease monitoring.
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Mentions: In order to determine if cytoplasmic TDP-43 aggregates can be detected in ALS-TES, 7-μm thick tissue sections were prepared and stained with commercial TDP-43 polyclonal antibody. Interestingly, TDP-43 cytoplasmic aggregates, characteristic of ALS pathology, were detected in SALS-derived skins by indirect immunofluorescence and standard microscopy (Figure 2). These results were also further confirmed by confocal microscopy, using 25-um thick sections (Additional file 5: Figure S3). To our knowledge, it is the first time that cytoplasmic TDP-43 aggregates are detected outside of the nervous system and in non-neuronal cells in any model so far. In contrast, no TDP-43 abnormal cytoplasmic accumulation was observed in control-derived reconstructed skin. To further confirm our results and determine if cytoplasmic TDP-43 can also be detected in non-symptomatic patients, we have generated C9orf72 FALS-derived TES. Five out of six generated C9orf72-TES were derived from non-symptomatic patients carrying the GGGGCC DNA repeat expansion (Additional file 2: Figure S1; Table 1). Remarkably, cytoplasmic TDP-43 inclusions were detected by standard immunofluorescence analysis in both symptomatic and yet non-symptomatic C9orf72-linked ALS patients carrying the expansion (Figure 2). Actually, around 30% of the fibroblasts within the C9orf72- and SALS-derived skins presented cytoplasmic TDP-43 positive inclusions while only 4% of the fibroblasts in the control-derived skins demonstrated TDP-43 cytoplasmic inclusions (Figure 3A). Validation of these results were done by Western blotting after proper fractionation of the cytoplasmic and nulear fractions (Figure 3B; Additional file 6: Figure S4). Interestingly, cytoplasmic TDP-43 inclusions were only detected in our three dimensional (3D) ALS-TES model and were not detected in patient’s fibroblasts alone standard two dimensional (2D) cell culture indicating that our 3D skin model is necessary to observe the described phenotype (Figure 4). Western immunoblots, detected with nuclear (anti-nuclei antibody, clone 235-1, Millipore: cat# MAB1281) and cytoplasmic (anti-GAPDH antibody, AbD Serotec: cat# AHP1628) markers, revealed that our cytoplasmic fraction was completely free of nuclear protein indicating that the detected cytolasmic TDP-43 signal was not due to a contamination of the fraction with nuclear proteins (Additional file 6: Figure S4).Figure 2