cells Article TDP-43 Cytoplasmic Translocation in the Skin Fibroblasts of ALS Patients Miguel A. Rubio 1,2 , Mireia Herrando-Grabulosa 2 , Roser Velasco 2,3 , Israel Blasco 2 , Monica Povedano 4 and Xavier Navarro 2, * 1 Neuromuscular Unit, Department of Neurology, Hospital del Mar, 08003 Barcelona, Spain; marubio@psmar.cat 2 Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; mireia.herrando@uab.cat (M.H.-G.); francast2@msn.com (R.V.); israel.blasruan@gmail.com (I.B.) 3 Neuro-Oncology Unit, Department of Neurology, Hospital Universitari de Bellvitge-ICO and IDIBELL, 08907 L’Hospitalet, Spain 4 Department of Neurology, Hospital Universitari de Bellvitge, 08907 L’Hospitalet, Spain; 30058mpp@gmail.com * Correspondence: xavier.navarro@uab.cat; Tel.: +34-935811966; Fax: +34-935812986 Abstract: Diagnosis of ALS is based on clinical symptoms when motoneuron degeneration is sig- nificant. Therefore, new approaches for early diagnosis are needed. We aimed to assess if alter- ations in appearance and cellular localization of cutaneous TDP-43 may represent a biomarker for ALS. Skin biopsies from 64 subjects were analyzed: 44 ALS patients, 10 healthy controls (HC) and 10 neurological controls (NC) (Parkinson’s disease and multiple sclerosis). TDP-43 immunoreactivity in epidermis and dermis was analyzed, as well as the percentage of cells with TDP-43 cytoplasmic  localization. We detected a higher amount of TDP-43 in epidermis (p < 0.001) and in both layers of  dermis (p < 0.001), as well as a higher percentage of TDP-43 cytoplasmic positive cells (p < 0.001) in Citation: Rubio, M.A.; the ALS group compared to HC and NC groups. Dermal cells containing TDP-43 were fibroblasts as Herrando-Grabulosa, M.; Velasco, R.; identified by co-labeling against vimentin. ROC analyses (AUC 0.867, p < 0.001; CI 95% 0.800–0.935) Blasco, I.; Povedano, M.; Navarro, X. showed that detection of 24.1% cells with cytoplasmic TDP-43 positivity in the dermis had 85% TDP-43 Cytoplasmic Translocation in sensitivity and 80% specificity for detecting ALS. We have identified significantly increased TDP-43 the Skin Fibroblasts of ALS Patients. levels in epidermis and in the cytoplasm of dermal cells of ALS patients. Our findings provide Cells 2022, 11, 209. https://doi.org/ 10.3390/cells11020209 support for the use of TDP-43 in skin biopsies as a potential biomarker. Academic Editor: Giuseppe Keywords: amyotrophic lateral sclerosis; dermis; skin biopsy; TDP-43; biomarker Marangi Received: 3 December 2021 Accepted: 4 January 2022 Published: 8 January 2022 1. Introduction Publisher’s Note: MDPI stays neutral Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor with regard to jurisdictional claims in neurons from cortex, brainstem, and spinal cord. While most are sporadic cases, 10–15% are published maps and institutional affil- familial forms [1,2]. Studies derived from the genetic forms have expanded the spectrum iations. of the disease to extra-motor manifestations such as cognitive impairment, extrapyrami- dal or neuropsychiatric symptoms [3–6]. Diagnosis is based on clinical symptoms and often is established relatively late; therefore, there is a need to identify biomarkers for the early diagnosis of ALS that could allow for monitoring disease progression and to start Copyright: © 2022 by the authors. early neuroprotective treatment to prevent the motoneuron degeneration. The 43-kDa Licensee MDPI, Basel, Switzerland. TAR DNA-binding protein (TDP-43) is a ubiquitous DNA binding protein with multiple This article is an open access article functions encoded by the TARDBP gene, and its mutations have been associated with distributed under the terms and autosomal dominant ALS and frontotemporal dementia (FTD) [7,8]. However, the more conditions of the Creative Commons than 50 missense mutations identified in TARDBP only account for 1–2% of total ALS Attribution (CC BY) license (https:// cases [9]. Nevertheless, the importance of TDP-43 in ALS relies on the fact that it is a major creativecommons.org/licenses/by/ component of the ubiquitinated insoluble cytoplasmic inclusions, concomitant with a loss 4.0/). Cells 2022, 11, 209. https://doi.org/10.3390/cells11020209 https://www.mdpi.com/journal/cells Cells 2022, 11, 209 2 of 12 of nuclear TDP-43 in upper and lower motor neurons and in other regions of the central nervous system in most patients (both sporadic and familial, with or without TARDBP mutations) [10]. These inclusions are widespread, regardless of the location of symptoms onset, and considered a pathological hallmark of ALS-FTD spectrum [11,12]. In healthy motoneurons, the TDP-43 protein is located in the nucleus, suggesting that the increased TDP-43 cytoplasmic translocation may be a potential pathological biomarker. However, monitoring this alteration using biopsies from the central nervous system is unpractical. Nevertheless, alterations in the skin and other tissues may precede or appear concomi- tantly with neurological symptoms in some neurodegenerative diseases [13]. Despite the variability among the studies performed on either skin biopsies, cultured fibroblasts or engineered skin tissue, there is a common finding of TDP-43 cytoplasmic accumulation in the skin of sporadic and familial ALS patients [14–21]. Nonetheless, there is not a well- defined relationship with disease progression and other clinical features. Moreover, nuclear TDP-43 expression is found in skin cells, fibroblasts, keratinocytes, Langerhans cells and melanocytes of healthy individuals, but importantly, much lower amounts are found in the cytoplasm [22,23]. Therefore, although cutaneous TDP-43 seems a promising candidate as a minimally invasive biomarker of the disease, its role is still undefined [22]. Quantitative detailed studies are needed to define the role of skin TDP-43 and its relationship with clinical features. The objective of this study was to further investigate the cytoplasmic localization of TDP-43 in the skin of ALS patients, quantifying its accumulation to identify more accessible histopathological hallmarks of ALS. Our results show an increase in TDP-43 in ALS patients in both epidermis and dermis, establishing a proof-of-concept of its possible value in the diagnosis of this pathology. 2. Materials and Methods 2.1. Participants and Samples Forty-four subjects with definite ALS and 10 healthy controls (HC) were prospectively recruited by the ALS units from Hospital del Mar and Hospital de Bellvitge, Barcelona. A third group of 10 neurological controls (NC) were also selected. ALS patients fulfilled the diagnostic criteria (revised El Escorial criteria) [24] and were selected by experienced neurologists specialized in motor neuron diseases. ALS patients were 22 females and 22 males, with a median age of 66 years (IQR 58–73). Healthy subjects did not have any signs, symptoms, or history of neurological diseases; 6 were female, with a median age of 59 years (IQR 43–62). The NC group consisted of 5 patients with Parkinson disease and 5 with relapsing-remitting multiple sclerosis, 4 of them were female, and had a median age of 59 years (IQR 54–66). There were no statistically significant differences in sociodemographic variables between the groups. Clinical data such as site of onset, time from onset of the disease to the biopsy and ALSFRS-R slope were obtained for the ALS group. Of the ALS patients, 18 (40.9%) had spinal onset and 26 bulbar onset (59.1%), and the median ALSFR-R slope at the time of the skin biopsy was 1.29 (IQR 0.63–2.18). Median time from onset of ALS symptoms to biopsy was 11.5 months (IQR 8.52–19.13). All ALS patients were tested for the hexanucleotide expansion in the C9ORF72 gene, and in all but one, no pathological expansion was detected. The study was approved by the local ethics committee of both participating hospitals. Written informed consent for skin biopsy was obtained from all subjects. Any method detail and data not published within the article will be shared anonymized by reasonable request from any qualified investigator. The samples were shipped to our laboratory coded, and the experimenters performing processing and immunohistochemical analyses were blind with respect to the diagnosis. 2.2. Skin Biopsy and Processing Three-millimeter skin punch biopsies were obtained from the distal leg after local anesthesia with mepivacaine. Skin biopsies were fixed in paraformaldehyde solution at 4% and maintained at 4 ◦ C in PB (phosphate buffer) with sucrose. Then, biopsies were cut by cryotome into 60 µm thick sections collecting the slices in PBS. Cells 2022, 11, x FOR PEER REVIEW 3 of 12 4% and maintained at 4 °C in PB (phosphate buffer) with sucrose. Then, biopsies were cut Cells 2022, 11, 209 3 of 12 by cryotome into 60 µ m thick sections collecting the slices in PBS. For immunofluorescence, sections were blocked with endogenous Biotin-Blocking Kit (Invitrogen), TBS-Triton 0.3% and normal donkey serum 10%, and subsequently incu- batedFor immunofluorescence, overnight sections were at 4 °C with anti-TDP-43 blocked(1:200, antibody with endogenous cat#12892-1-AP, Biotin-Blocking Proteintech, Kit (Invitrogen), Manchester, UK),TBS-Triton anti-vimentin 0.3% and normal (1:200, Sigma, St. donkey Louis,serum MO, USA)10%,andandrabbit subsequently in- anti-protein cubated overnight gene product at 4 ◦ C with 9.5 (PGP9.5, 1:50,anti-TDP-43 Cedarlane, antibody Burlington, (1:200, cat#12892-1-AP, Canada) as primaryProteintech, antibodies. Manchester, After washes, UK), anti-vimentin sections (1:200, Sigma, were incubated St. Louis, overnight at 4 °CMO, USA) with andanti-rabbit horse rabbit anti-protein biotinyl- gene ated antibody (1:200, Vector Laboratories, Burlingame, California, USA), antibodies. product 9.5 (PGP9.5, 1:50, Cedarlane, Burlington, Canada) as primary conjugated After washes,Alexa streptavidin sections were488 Fluor incubated at 4 ◦ CWaltham, overnightFisher, (1:200, Thermo with horseMassachusetts, anti-rabbit biotinylated USA) or antibody (1:200, streptavidin Vector Alexa Laboratories, Fluor 594 (1:200,Burlingame, Thermo Fisher, California, Waltham, USA), conjugated streptavidin Massachusetts, USA) and Alexa donkey Fluor 488 (1:200, anti-rabbit Thermo cyanine Fisher,Jackson 3 (1:200, Waltham, Massachusetts, USA) Immunoresearch, or streptavidin Alexa Ely, Cambridgeshire, UK) Fluor 594 (1:200, Thermo Fisher, Waltham, Massachusetts, USA) and as secondary antibodies. Slices were then transferred to gelatinized slides and mounted donkey anti-rabbit cyanine 3 (1:200,GJackson in Fluoromount Immunoresearch, (SouthernBiotech, Ely, Cambridgeshire, Birmingham, AL, USA). To assessUK) as secondary antibody anti- specific- bodies. Slices were then transferred to gelatinized slides and mounted ity, control samples were processed in parallel as described before but without primary in Fluoromount G (SouthernBiotech, Birmingham, AL, USA). To assess antibody specificity, antibodies. DAPI staining was used to ensure correct recognition of tissue structures. The control samples were processed anti-TDP43 in parallel antibody usedasindescribed this study before but without recognizes the C primary terminalantibodies. DAPI stain- cleavage product (20– ing was used to ensure correct recognition of tissue structures. The 30 KDa) and the native and phosphorylated forms of TDP-43. Antibodies against the non- anti-TDP43 antibody used in this studyform phosphorylated recognizes have been thewidely C terminal usedcleavage product to determine the (20–30 TDP-43KDa) and the native translocation to the and phosphorylated forms of TDP-43. Antibodies against the non-phosphorylated form cytoplasm without the risk of missing different post-translational modifications and non- have been widely used to determine the TDP-43 translocation to the cytoplasm without the phosphorylated cytosolic distribution of TDP-43 [12,16,26]. In the studies published to risk of missing different post-translational modifications and non-phosphorylated cytosolic date on skin of ALS patients, only two have used antibodies against p-TDP-43, finding no distribution of TDP-43 [12,16,25]. In the studies published to date on skin of ALS patients, labeling in one of them [27] and labeling only one-third of the cases in the other [28]. The only two have used antibodies against p-TDP-43, finding no labeling in one of them [26] rest of the studies have successfully used antibodies against the non-phosphorylated form and labeling only one-third of the cases in the other [27]. The rest of the studies have [14–21]. successfully used antibodies against the non-phosphorylated form [14–21]. 2.3. Confocal 2.3. Confocal Imaging Imaging and and Measurements Measurements Immunolabeled sections Immunolabeled sections were were first first viewed viewed under under anan Olympus Olympus BX-51 BX-51 microscope microscope equipped for epifluorescence using appropriate filters. Areas of interest were analyzed equipped for epifluorescence using appropriate filters. Areas of interest were analyzed with aa scanning with scanning confocal confocal microscope microscope(Figure (Figure1). 1). Figure 1. Figure 1. Cytoplasmic Cytoplasmic TDP-43 immunoreactivity was analyzed in at least two areas of epidermis, papillary and papillary and reticular reticular dermis dermis (dotted (dotted rectangular rectangular area), area), per per case case (left (left panel). panel). In In the the right right panel, panel, representative confocal microscopy images of epidermis (A), papillary dermis (B) and reticular representative confocal microscopy images of epidermis (A), papillary dermis (B) and reticular dermisder- mis (C) from ALS, HC and NC subjects are shown. TDP-43 was found profusely in epidermis in all (C) from ALS, HC and NC subjects are shown. TDP-43 was found profusely in epidermis in all cases, cases, although with higher density in the ALS group (p < 0.001) compared to both HC and NC although with higher density in the ALS group (p < 0.001) compared to both HC and NC groups. In groups. In the dermis of HC and NC subjects, TDP-43 was mostly seen in the nucleus, whereas the dermis of HC and NC subjects, TDP-43 was mostly seen in the nucleus, whereas cytoplasmic aggregations were more present in ALS cases in papillary and reticular dermis (p < 0.001). ALS: amyotrophic lateral sclerosis; HC: healthy controls; NC: neurological controls. Cells 2022, 11, 209 4 of 12 Epidermal TDP-43 amount was measured in microphotographs taken from two repre- sentative areas of epidermis (101.61 × 101.61 µm2 each) of each case, using ImageJ software (version 2.1.0/1.53c, Wayne Rasband, Bethesda, Maryland, USA). The mean of the two measures of the percentage of area with TDP-43 labeling was calculated after defining the threshold background correction. The two layers of the dermis (papillary and reticular dermis) were evaluated sepa- rately. This distinction was made given that papillary and reticular cells have different patterns of protein synthesis and expression [25,28]. Differentiation of both layers was based on visual recognition of different density of connective tissue, and the superficial vascular plexus at the boundaries between both layers. Images of two representative areas (101.61 × 101.61 µm2 each) of papillary and reticular dermis were taken and analyzed. The representative areas were selected in a systematic way, avoiding areas of the section that might have artefacts and then selecting 2 evenly separated fields (for epidermis, papillary and reticular dermis) at low magnification, and then viewing them under higher magni- fication for capture and analysis. The levels of cytoplasmic TDP-43 were quantified as (1) percentage of cytoplasmic TDP-43 immunoreactivity and (2) percentage of cells with positive TDP-43 into the cytoplasm in each defined area. For calculating the percentage of cytoplasmic TDP43 immunoreactivity, first, a threshold was defined for background correction, then the percentage of pixels in the area above threshold of TDP-43 labeling was measured using ImageJ software. Additionally, the mean percentage of cells with TDP-43 positivity within their cytoplasm was calculated in the analyzed confocal images, relative to the total number of vimentin positive cells (fibroblasts) in these areas. Results are expressed as the mean of the two measures per subject and layer. 2.4. Intraepidermal Innervation and TDP-43 Nerve Colocalization To study the possible colocalization of TDP-43 in the cutaneous innervation, we performed immunohistochemical co-labeling for PGP9.5 and TDP-43. Confocal images were taken with a confocal microscope for counting intraepidermal nerve fibers (IENF) and for TDP-43 colocalization assessment. IENF were measured in a subset of ALS and HC subjects. Individual fibers were counted as they pass through the basement membrane, whereas branching occurring within the epidermis did not increase the number of the IENFs counted. IENF density was expressed as the number of fibers per 1 mm length of the epidermis. Average density of IENFs in two images per sample was then derived. 2.5. Data Analysis Data are expressed as mean ± SEM. Means were compared by ANOVA, applying Tukey’s post hoc test when necessary (SPSS statistics 19 software, IBM, Armonk, NY, USA). The level of significance was set at p < 0.05. Pearson’s correlation coefficient was used to assess possible linear association between two continuous quantitative variables. In order to obtain an estimate of sensitivity and specificity of TDP-43 quantification regarding ALS diagnosis, ROC curves and area under the curve (AUC) were calculated and optimal cut-off values were selected using Youden’s index. 3. Results 3.1. TDP-43 Cytoplasmic Localization in ALS Patients Skin Biopsies To determine cytoplasmic TDP-43 accumulation, a signature of ALS pathology, skin biopsies, were labeled with polyclonal antibody against TDP-43. The results of TDP-43 immunoreactivity levels and proportion of cells with cytoplasmic TDP-43 positivity in epidermis, papillary and reticular dermis are summarized in Table 1. Cells 2022, 11, 209 5 of 12 Cells 2022, 11, x FOR PEER REVIEW Table 1. Results of TDP-43 immunoreactivity and cells with cytoplasmic TDP-43-positive 5 ofcells. 12 Values are shown as mean and SEM. ALS: amyotrophic lateral sclerosis; HC: healthy controls; NC: neurological controls (5 multiple sclerosis and 5 Parkinson’s disease); IR: immunoreactivity. Table 1. Results of TDP-43 immunoreactivity and cells with cytoplasmic TDP-43-positive cells. Val- ues are shown as meanALS and(N = 44) SEM. HC (N = 10)lateral sclerosis; ALS: amyotrophic NC (N =HC: p Value NC: 10) healthy controls; neurological controls (5 multiple sclerosis and 5 Parkinson’s disease); IR: immunoreactivity. Epidermis % TDP-43 IR 12.94 ± 1.05 7.19 ± 0.97 4.37 ± 1.25 <0.001 ALS (N = 44) HC (N = 10) NC (N = 10) p Value Papillary Epidermis Dermis % TDP-43 % TDP-43 IR IR 1.33 ± 0.17 12.94 ±0.13 1.05± 0.057.19 ± 0.97 4.37 ± 1.25 0.31 ± 0.07 <0.001 <0.001 % Cytoplasmic Papillary Dermis + cells 50.88 ± 3.91 19.11 ± 5.2 16.58 ± 4.85 <0.001 TDP-43% TDP-43 IR 1.33 ± 0.17 0.13 ± 0.05 0.31 ± 0.07 <0.001 Reticular % Cytoplasmic TDP-43+ cells 50.88 ± 3.91 19.11 ± 5.2 16.58 ± 4.85 <0.001 Dermis Reticular Dermis % TDP-43 IR 0.82 ± 0.09 0.18 ± 0.07 0.10 ± 0.03 <0.001 % TDP-43 IR % Cytoplasmic 0.82 ± 0.09 0.18 ± 0.07 0.10 ± 0.03 <0.001 % TDP-43 Cytoplasmic 50.36 + cells TDP-43 ± 3.64 50.36 ±21.73 + cells 3.64± 6.40 11.75 ± 11.75 21.73 ± 6.40 5.88 ± 5.88 <0.001 <0.001 In epidermis, TDP-43 was distributed in stratum basale and stratum spinosum. Ab- In epidermis, TDP-43 was distributed in stratum basale and stratum spinosum. Ab- sence of TDP-43 in the outer layers of the skin may be explained by the process of terminal sence of TDP-43 in the outer layers of the skin may be explained by the process of terminal differentiation of keratinocytes in granulosum and corneum strata. We detected a higher differentiation of keratinocytes in granulosum and corneum strata. We detected a higher ex- expression of TDP-43 in epidermal cells (p < 0.001) in the ALS group (12.9 ± 1.0%) com- pression of TDP-43 in epidermal cells (p < 0.001) in the ALS group (12.9 ± 1.0%) compared pared to HCto HC±(7.2 (7.2 ± 1.0%) 1.0%) and(4.4 and NC NC±(4.4 ± 1.2%) 1.2%) (Figure (Figure 2). 2). Figure 2. Distribution of the amount of cytoplasmic TDP-43 content and cells with cytoplasmic Figure 2. Distribution of the amount of cytoplasmic TDP-43 content and cells with cytoplasmic TDP- TDP-43 of the three cohorts in the different skin layers. IR: immunoreactivity; ALS: amyotrophic 43 of the three cohorts in the different skin layers. IR: immunoreactivity; ALS: amyotrophic lateral lateral sclerosis; HC: healthy controls; NC: neurological controls. sclerosis; HC: healthy controls; NC: neurological controls. Cytoplasmic TDP-43 was detected in dermal cells of ALS patients and controls. How- Cytoplasmic TDP-43 was detected in dermal cells of ALS patients and controls. How- ever, a higher number of positive cells for cytosolic labeling of TDP-43 was found in ALS ever, a higher number of positive cells for cytosolic labeling of TDP-43 was found in ALS patients than in healthy and NC. Those dermal cells were identified as fibroblasts as they patients than in healthy were positively markedandwithNC. Those (Figure vimentin dermal 3).cells were what Unlike identified as fibroblasts was seen as they in the epidermis, were positively marked with vimentin (Figure 3). Unlike what was seen in the fibroblasts expressing TDP-43 were not widely distributed, and represented only a small epidermis, fibroblasts percentageexpressing of dermal TDP-43 cells. In were notcells, dermal widely distributed, TDP-43 and represented cytoplasmic distributiononly a small varied from percentage of dermal cells. In dermal cells, TDP-43 cytoplasmic distribution varied small amounts around the nucleus to a more scattered deposition, whereas in the epidermis, from small thoseamounts aggregatesaround were the morenucleus dense to anda more evenly scattered diffuseddeposition, within the whereas cells. Theinimmunoreac- the epider- mis, those aggregates were more dense and evenly diffused within the cells. tivity against TDP-43 as well as the percentage of fibroblast cells with cytoplasmic The immu- label noreactivity against TDP-43 as well as the percentage of fibroblast cells with for TDP-43 were significantly increased in the ALS group compared with the HC and NC cytoplasmic label groupsforinTDP-43 were both the significantly papillary increased and reticular dermisin the ALS1).group compared with the HC (Table and NC groups in both the papillary and reticular dermis (Table 1). Cells 2022, 11,2022, Cells 209 11, x FOR PEER REVIEW 6 of 126 of 12 Figure Figure 3. CytoplasmicTDP-43-positive 3. Cytoplasmic TDP-43-positive cells (*) (*) cells were identified were as fibroblasts identified based on as fibroblasts vimentin based im- on vimentin munoreactivity. Representative confocal microscopy images of ALS dermal fibroblasts co-immuno- immunoreactivity. Representative confocal microscopy images of ALS dermal fibroblasts co- labeled for TDP-43 (in green), vimentin (in red) and DAPI nuclear staining (in blue). immunolabeled for TDP-43 (in green), vimentin (in red) and DAPI nuclear staining (in blue). We performed an ROC analysis based on the percentage of TDP-43 cytoplasmic-pos- Wecells itive performed an ROC of the overall dermisanalysis as well asbased on the on the percentage percentage of TDP-43tocytoplasmic- of immunoreactivity achieve positive a theoretical cut-off point as a proof of concept of its value as a markerofofimmunoreactivity cells of the overall dermis as well as on the percentage the disease. ROC to achieve a theoretical cut-off point as a proof of concept of its value as curves and AUC (0.867, p < 0.001; CI 95% 0.800–0.935) were calculated for TDP-43-positive a marker of the disease. dermalROC cells,curves and including AUC (0.867, measures p < 0.001; CI dermis, from superficial/deep 95% 0.800–0.935) were calculated and cut-off values were for TDP-43-positive selected according dermal cells, to optimal including Youden’s index.measures Detectionfrom superficial/deep of 24.14% dermis, and of cells with cytoplas- mic values cut-off TDP-43werepositivity in the selected dermis had according 85.2% sensitivity to optimal Youden’s and 80.0%Detection index. specificity,ofwith a 24.14% of positive predictive value of 90.4% and a negative predictive value of cells with cytoplasmic TDP-43 positivity in the dermis had 85.2% sensitivity and 80.0% 71.1% (Figure 4). Considering specificity, withthe proportion a positive of TDP-43 predictive immunoreactivity value of 90.4% andinathe dermis,predictive negative ROC analyses value of showed an AUC of 0.911 (p < 0.001, CI 95% 0.862–0.960) and 71.1% (Figure 4). Considering the proportion of TDP-43 immunoreactivity cut-off values of 0.26%, in thewith dermis, 88.6% sensitivity, 82.5% specificity, 91.8% positive predictive value and 76.7% negative ROC analyses showed an AUC Cells 2022, 11, x FOR PEER REVIEWpredictive value for detecting ALS. of 0.911 (p < 0.001, CI 95% 0.862–0.960) and cut-off values 7 of 12 of 0.26%, with 88.6% sensitivity, 82.5% specificity, 91.8% positive predictive value and 76.7% There were no differences in TDP-43 levels (immunoreactivity and percentage of cells negative predictive TDP-43 with cytoplasmic value for detecting positivity) ALS. in epidermis and papillary dermis according to gen- der and site of onset in ALS patients. Only in the reticular dermis we found a significant lower TDP-43 immunoreactivity in bulbar onset patients, but no differences were found considering the percentage of cells with cytoplasmic TDP-43 localization. No significant correlations were found between TDP-43 expression measurements and age of onset of the disease, time from symptoms onset to biopsy and ALSFRS-R slope (Table 2; Supple- mental Figure S1). Figure 4. ROC Figure curves 4. ROC of of curves thethe percentage percentageof of TDP-43 immunoreactivity TDP-43 immunoreactivity in in both both layers layers of dermis of the the dermis (AUC(AUC 0.911, CI 95% 0.911, CI 95% 0.862–0.960,p p< <0.001) 0.862–0.960, 0.001) and and of thethe percentage percentageofof dermal dermal cells cells withwith cytoplasmic cytoplasmic TDP-43 TDP-43 labeling labeling (AUC(AUC 0.867, 0.867, CICI 95%95%0.800–0.935, 0.800–0.935, pp << 0.001) 0.001)ininALS ALSpatients. ROC: patients. receiver ROC: operation receiver operation characteristic; AUC: area under the ROC curve. characteristic; AUC: area under the ROC curve. Table 2. TDP-43 quantification and clinical features of ALS patients (N = 44). IR: immunoreactivity; R: Pearson correlation coefficient. Epidermis Superficial Dermis Deep Dermis % Cytoplasmic TDP-43+ % Cytoplasmic TDP-43+ Cells 2022, 11, 209 7 of 12 There were no differences in TDP-43 levels (immunoreactivity and percentage of cells with cytoplasmic TDP-43 positivity) in epidermis and papillary dermis according to gender and site of onset in ALS patients. Only in the reticular dermis we found a significant lower TDP-43 immunoreactivity in bulbar onset patients, but no differences were found considering the percentage of cells with cytoplasmic TDP-43 localization. No significant correlations were found between TDP-43 expression measurements and age of onset of the disease, time from symptoms onset to biopsy and ALSFRS-R slope (Table 2; Supplemental Figure S1). Table 2. TDP-43 quantification and clinical features of ALS patients (N = 44). IR: immunoreactivity; R: Pearson correlation coefficient. Epidermis Superficial Dermis Deep Dermis % Cytoplasmic % Cytoplasmic % TDP-43 IR % TDP-43 IR % TDP-43 IR TDP-43+ Cells TDP-43+ Cells Gender male female 14.66 ± 1.33 1.24 ± 0.19 47.37 ± 5.60 0.85 ± 0.12 50.96 ± 5.34 11.21 ± 1.57 1.43 ± 0.29 54.39 ± 5.49 0.81 ± 0.13 47.95 ± 4.50 p = 0.102 p = 0.601 p = 0.376 p = 0.832 p = 0.669 Site of onset spinal bulbar 14.31 ± 1.21 1.33 ± 0.20 46.57 ± 5.30 0.98 ± 0.12 49.02 ± 4.40 10.52 ± 1.86 1.34 ± 0.34 58.43 ± 5.12 0.55 ± 0.08 50.21 ± 5.76 p = 0.083 p = 0.991 p = 0.147 p = 0.017 p = 0.871 R = −0.050 R = −0.180 R = −0.199 R = 0.043 R = −0.114 Age p = 0.747 p = 0.243 p = 0.194 p = 0.780 p = 0.460 Time from onset R = 0.071 R = 0.032 R = −0.115 R = 0.053 R = −0.119 to biopsy p = 0.646 p = 0.836 p = 0.457 p = 0.731 p = 0.440 R = −0.169 R = −0.205 R = −0.046 R = −0.086 R = 0.057 ALSFRS-R slope p = 0.277 p = 0.186 p = 0.772 p = 0.585 p = 0.717 3.2. TDP-43 Changes over Time A small subset of ALS patients (eight in total; three bulbar and five spinal onset) underwent a second skin biopsy 12 months later. There were no significant differences in levels of TDP-43 immunoreactivity in epidermis (17.29 ± 5.33 vs. 18.58 ± 3.68; p = 0.831), superficial dermis (1.17 ± 0.34 vs. 8.58 ± 6.24; p = 0.283) and deep dermis (3.85 ± 3.17 vs. 16.29 ± 11.32; p = 0.336) between the initial biopsy and 12 months later. The increase in levels of TDP-43 in the dermis at the second biopsy did not achieve statistical significance due to the low number of samples analyzed. However, the percentage of cells with cytoplasmic TDP-43 translocation in the dermis increased in the second biopsy, with differences being significant for the papillary dermis (34.82 ± 7.79 vs. 62.13 ± 3.82; p = 0.034), but not for the reticular dermis (49.73 ± 10.01 vs. 62.38 ± 2.32; p = 0.241). 3.3. TDP-43 and Epidermal Nerve Fibers We analyzed the IENF density of 18 ALS patients and six controls. The ALS group presented lower values of IENF (7.78 ± 0.80; p = 0.010) compared with HC (12.75 ± 2.20) and NC (10.89 ± 1.77). However, no colocalization of TDP-43 was found in PGP9.5-labeled fibers in ALS patients, HC or NC (Figure 5). but not for the reticular dermis (49.73 ± 10.01 vs. 62.38 ± 2.32; p = 0.241). 3.3. TDP-43 and Epidermal Nerve Fibers We analyzed the IENF density of 18 ALS patients and six controls. The ALS group presented lower values of IENF (7.78 ± 0.80; p = 0.010) compared with HC (12.75 ± 2.20) Cells 2022, 11, 209 and NC (10.89 ± 1.77). However, no colocalization of TDP-43 was found in PGP9.5-labeled8 of 12 fibers in ALS patients, HC or NC (Figure 5). Figure Figure 5. Representative 5. Representative images images ofofALS ALSskin skinsections sections immunohistochemically immunohistochemically labeled forfor labeled PGP 9.59.5 (in PGP (in red) to identify the innervation and for TDP-43 (in green). In the images, subepidermal red) to identify the innervation and for TDP-43 (in green). In the images, subepidermal nerve plexus nerve plexus and intraepidermal nerve fibers are shown with no colocalization of TDP-43 immunoreac- and intraepidermal nerve fibers are shown with no colocalization of TDP-43 immunoreactivity. tivity. 4. Discussion 4. Discussion The results of this study show that cytosolic localization of TDP-43 is increased in The results of this study show that cytosolic localization of TDP-43 is increased in the the skin skincells cellsofofALS ALS patients patients andandcan can be detected be detected by immunofluorescence by immunofluorescence of skin biop- of skin biopsies. sies.TDP-43 TDP-43 presence in the cytoplasm of dermal fibroblasts is particularly relevant and rep- and presence in the cytoplasm of dermal fibroblasts is particularly relevant represents a potentiallyuseful resents a potentially useful biomarker biomarker of ALS. of ALS. TDP-43 cytoplasmic deposition TDP-43 cytoplasmic deposition and its and itsclearance clearance from from thethe nucleus nucleus havehave been been ob- ob- served beyond served beyond motoneurons motoneurons in in other areasofofthe other areas thenervous nervous system, system, andand in extra-neural in extra-neural tissues tissues [19,30–34]. [19,29–33]. However,few However, few studies studies have haveexplored explored thethe presence presenceof TPD-43 of TPD-43protein in protein in skin of ALS patients, and quantitative information from the dermis skin of ALS patients, and quantitative information from the dermis of skin biopsies was of skin biopsies was missing. missing. Suzuki Suzuki et et al.al.[14] [14]analyzed analyzed skin skinbiopsies biopsiesfrom from 1515ALS patients ALS andand patients 15 neurological 15 neurological controls and found higher amounts of TDP-43 in the controls and found higher amounts of TDP-43 in the ALS group, although ALS group, although theythey did not did not report on the nuclear/cytoplasmic localization of the protein. They also observed TDP-43 report on the nuclear/cytoplasmic localization of the protein. They also observed TDP-43 immunoreactivity in both neurological controls and ALS in blood vessels and glands, but immunoreactivity in both neurological controls and ALS in blood vessels and glands, but it it was lower in the case of controls [31]. In a tissue-engineered model derived from fibro- wasblasts lowerofin12the case of controls [30]. In a tissue-engineered model derived from fibroblasts ALS patients (six sporadic and six familial C9ORF72 cases), an increased ac- of 12cumulation ALS patients (six sporadic of cytoplasmic and was TDP-43 six familial found inC9ORF72 ALS patients cases), (30%)ancompared increased toaccumulation HC (4%) of cytoplasmic in epidermis, TDP-43 was found junction, dermo-epidermal in ALS patients (30%) and dermis compared [14]. Wang etto al.HC[15](4%) in epidermis, observed in dermo-epidermal patients harboring junction, the TARDBPand dermis A315T[14]. Wang mutation et the that al. [15] observed presence in patients of cytoplasmic harboring TDP-43 the TARDBP A315T mutation that the presence of cytoplasmic TDP-43 occurs mainly in epidermis but also in dermis, although no quantification was performed. Another study with skin biopsies from 22 ALS patients and 26 neurological controls (neuropathies) [19] analyzed TDP-43 mRNA expression, epidermal TDP-43 immunoblot and percentage of TDP-43-positive cells in epidermis. They did not find differences in the immunohistochem- ical analysis, although differences in immunoblot TDP-43 expression with a reduction of mRNA in ALS epidermis suggested a dysregulation of protein expression. Studies on cultured fibroblasts have shown abnormal TDP-43 cytoplasmic aggrega- tions in ALS (sporadic and familial cases of SOD1, TARDBP, FUS and C9ORF72 muta- tions) [17], even with different patterns regarding nuclear/cytoplasmic deposition ratio between patients harboring different mutations. Other studies have focused on the cyto- plasmic aggregation in cultured fibroblasts under the presence of stress in both sporadic and familial cases (UBQLN2, UBQLN1, TARDBP) [18,20,21]. Only one study to date based on cultured fibroblasts from sporadic ALS patients did not detect any TDP-43 accumula- Cells 2022, 11, 209 9 of 12 tion [26]. Authors speculated that methodological aspects could explain differences from other similar studies. TDP-43 accumulation is not homogenous throughout the motor nervous system of ALS patients [12]. Full-length protein can be found in both cortical and spinal motor neurons, but C-terminal fragments have only been observed in brain neurons [34,35]. These differences in affected neurons raise the question if TDP-43 proteinopathy may be heterogeneous also in non-neuronal cells [36]. As expected, based on previous observations [37], we found a lower IENF density in the ALS group, but we did not find any colocalization of TDP-43 in dermal or intraepidermal nerve fibers. To date, only deposition of TDP-43 in autonomic dermal fibers has been shown [27]. We did not find a correlation between TDP-43 deposition and clinical data. There are only few previous reports about clinical correlation, and only disease duration was analyzed with contradictory results. In the subgroup of patients with a second biopsy 12 months later, there was a high variability of TDP-43 immunoreactivity between the first and second biopsies. Although changes were not significant, a higher number of dermal cells with cytoplasmic TDP-43 deposition were detected in the superficial dermis. However, the conclusions from this subgroup should be taken with caution due to the small number of patients, and the limited time interval between biopsies in a fast-progression disease such as ALS. Albeit having some distinctive histopathological features, ALS lacks an accessible biomarker. Diagnosis is based on clinical criteria and supported by neurophysiological studies [24,38,39]. Although these criteria have been revised to achieve an early diagnosis with proper sensitivity and specificity, the time from symptoms onset to diagnosis ranges from 8 to 16 months, and even a non-negligible proportion of patients die without achieving a sufficient diagnostic certainty [40,41]. Even in the absence of a curative treatment, the benefits of a biomarker should not be overlooked, whether it is for the possibility of an earlier diagnosis in suspected cases, presymptomatic detection and an optimal selection of candidates for clinical trials. For this reason, determination of TDP-43 in corporal fluids or tissue as a potential biomarker has been pursued [42]. Different approaches have been taken for the detection and quantification of TDP-43 in blood [43–45] and cerebrospinal fluid (CSF) in ALS and FTD [46–49]. However, the ubiquitous nature of the TDP-43 protein makes its detection and interpretation more difficult compared to the detection of amyloid or tau (purely neuronal proteins) in Alzheimer’s disease [50]. TDP-43 cytoplasmic localization is present in epidermal keratinocytes and in dermal fibroblasts of ALS and HC, but quantitative analyses show significantly larger amounts in ALS patients. Quantitative values of dermal cells with TDP-43 appear to offer a sensitive and minimally invasive biomarker. Further experiments are needed to specifically explore the levels of post-translational modifications of TDP-43 in these samples, such as phospho- rylation, ubiquitylation and truncated forms. Although the number of skin samples from ALS patients represents the larger series reported to date, a higher amount is needed to achieve practical cut-off values of TDP-43 deposition for diagnostic purposes. Nevertheless, even considering the limitations, this work represents the proof of concept of cutaneous TDP-43 as a potential biomarker for ALS. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11020209/s1, Figure S1: Correlation graphs of the per- centage of cytoplasmic TDP-43 immunoreactivity with age, time from onset to biopsy and ALSFRS-R slope in the three different skin layers. Author Contributions: M.A.R. designed and conceptualized study, analyzed and interpreted the data, drafted the manuscript for intellectual content and performed sample and clinical data collection. M.H.-G. designed and conceptualized study, analyzed the data, interpreted the data and revised the manuscript for intellectual content. R.V. analyzed the data. I.B. processed the samples. M.P. performed sample and clinical data collection and revised the manuscript for intellectual content. X.N. designed and conceptualized the study, interpreted the data, drafted and revised the manuscript for intellectual content. All authors have read and agreed to the published version of the manuscript. Cells 2022, 11, 209 10 of 12 Funding: This work was supported by project RTI2018-096386-B-I00 from Ministerio de Ciencia, Inno- vación y Universidades and Agencia Española de Investigación of Spain, CIBERNED (CB06/05/1105) and TERCEL (RD16/0011/0014) funds from the Instituto de Salud Carlos III of Spain, co-funded by European Union (ERDF/ESF, “Investing in your future”). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Hospital del Mar (CEIC-PSMAR, approval code: 2015/6051/I, date of approval: 16-03-2015). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. Hardiman, O.; Al-Chalabi, A.; Chio, A.; Corr, E.M.; Logroscino, G.; Robberecht, W.; Van Den Berg, L.H. Amyotrophic lateral sclerosis. Nat. Rev. Dis. Primers 2017, 3, 1–19. [CrossRef] 2. Mancuso, R.; Navarro, X. Amyotrophic lateral sclerosis: Current perspectives from basic research to the clinic. Prog. Neurobiol. 2015, 133, 1–26. [CrossRef] 3. McCombe, P.A.; Wray, N.R.; Henderson, R.D. Extra-motor abnormalities in amyotrophic lateral sclerosis: Another layer of heterogeneity. Expert Rev. Neurother. 2017, 17, 561–577. [CrossRef] 4. McCluskey, L.; Vandriel, S.; Elman, L.; Van Deerlin, V.M.; Powers, J.; Boller, A.; Wood, E.M.; Woo, J.; McMillan, C.T.; Rascovsky, K.; et al. ALS-Plus syndrome: Non-pyramidal features in a large ALS cohort. J. Neurol. Sci. 2014, 345, 118–124. [CrossRef] 5. Strong, M.J. The syndromes of frontotemporal dysfunction in amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. 2008, 9, 323–338. [CrossRef] [PubMed] 6. Phukan, J.; Pender, N.; Hardiman, O. Cognitive impairment in amyotrophic lateral sclerosis. Lancet Neurol. 2007, 6, 994–1003. [CrossRef] 7. Kwong, L.K.; Neumann, M.; Sampathu, D.M.; Lee, V.M.-Y.; Trojanowski, J.Q. TDP-43 proteinopathy: The neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease. Acta Neuropathol. 2007, 114, 63–70. [CrossRef] 8. Neumann, M.; Sampathu, D.M.; Kwong, L.K.; Truax, A.C.; Micsenyi, M.C.; Chou, T.T.; Bruce, J.; Schuck, T.; Grossman, M.; Clark, C.M.; et al. Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science 2006, 314, 130–133. [CrossRef] [PubMed] 9. Chia, R.; Chiò, A.; Traynor, B.J. Novel genes associated with amyotrophic lateral sclerosis: Diagnostic and clinical implications. Lancet Neurol. 2018, 17, 94–102. [CrossRef] 10. Brettschneider, J.; Del Tredici, K.; Toledo, J.B.; Robinson, J.L.; Irwin, D.J.; Grossman, M.; Suh, E.R.; Van Deerlin, V.M.; Wood, E.M.; Baek, Y.; et al. Stages of pTDP-43 pathology in amyotrophic lateral sclerosis. Ann. Neurol. 2013, 74, 20–38. [CrossRef] 11. Neumann, M. Molecular Neuropathology of TDP-43 Proteinopathies. Int. J. Mol. Sci. 2009, 10, 232–246. [CrossRef] [PubMed] 12. Braak, H.; Ludolph, A.C.; Neumann, M.; Ravits, J.; Del Tredici, K. Pathological TDP-43 changes in Betz cells differ from those in bulbar and spinal α-motoneurons in sporadic amyotrophic lateral sclerosis. Acta Neuropathol. 2017, 133, 79–90. [CrossRef] [PubMed] 13. Clos, A.L.; Kayed, R.; Lasagna-Reeves, C.A. Association of Skin with the Pathogenesis and Treatment of Neurodegenerative Amyloidosis. Front. Neurol. 2012, 3, 5. [CrossRef] 14. Suzuki, M.; Mikami, H.; Watanabe, T.; Yamano, T.; Yamazaki, T.; Nomura, M.; Yasui, K.; Ishikawa, H.; Ono, S. Increased expression of TDP-43 in the skin of amyotrophic lateral sclerosis. Acta Neurol. Scand. 2010, 122, 367–372. [CrossRef] 15. Wang, X.; Zhou, S.; Ding, X.; Ma, M.; Zhang, J.; Zhou, Y.; Wu, E.; Teng, J. Activation of ER Stress and Autophagy Induced by TDP-43 A315T as Pathogenic Mechanism and the Corresponding Histological Changes in Skin as Potential Biomarker for ALS with the Mutation. Int. J. Biol. Sci. 2015, 11, 1140–1149. [CrossRef] 16. Paré, B.; Touzel-Deschênes, L.; Lamontagne, R.; Lamarre, M.-S.; Scott, F.-D.; Khuong, H.T.; Dion, P.A.; Bouchard, J.-P.; Gould, P.; Rouleau, G.A.; et al. Early detection of structural abnormalities and cytoplasmic accumulation of TDP-43 in tissue-engineered skins derived from ALS patients. Acta Neuropathol. Commun. 2015, 3, 1–12. [CrossRef] 17. Sabatelli, M.; Zollino, M.; Conte, A.; Del Grande, A.; Marangi, G.; Lucchini, M.; Mirabella, M.; Romano, A.; Piacentini, R.; Bisogni, G.; et al. Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations. Neurobiol. Aging 2015, 36, 2005-e5–2005-e13. [CrossRef] 18. Yang, S.; Zhang, K.Y.; Kariawasam, R.; Bax, M.; Fifita, J.A.; Ooi, L.; Yerbury, J.J.; Nicholson, G.A.; Blair, I.P. Evaluation of Skin Fibroblasts from Amyotrophic Lateral Sclerosis Patients for the Rapid Study of Pathological Features. Neurotox. Res. 2015, 28, 138–146. [CrossRef] Cells 2022, 11, 209 11 of 12 19. Abe, K.; Ohkubo, T.; Yokota, T. TDP-43 in the skin of amyotrophic lateral sclerosis patients. J. Med. Dent. Sci. 2017, 64, 9–17. [PubMed] 20. Riancho, J.; Castanedo-Vázquez, D.; Gil-Bea, F.; Tapia, O.; Arozamena, J.; Durán-Vían, C.; Sedano, M.J.; Berciano, M.T.; De Munain, A.L.; Lafarga, M. ALS-derived fibroblasts exhibit reduced proliferation rate, cytoplasmic TDP-43 aggregation and a higher susceptibility to DNA damage. J. Neurol. 2020, 267, 1291–1299. [CrossRef] 21. Romano, N.; Catalani, A.; Lattante, S.; Belardo, A.; Proietti, S.; Bertini, L.; Ceci, M. ALS skin fibroblasts reveal oxidative stress and ERK1/2-mediated cytoplasmic localization of TDP-43. Cell. Signal. 2020, 70, 109591. [CrossRef] [PubMed] 22. Uhlen, M.; Oksvold, P.; Fagerberg, L.; Lundberg, E.; Jonasson, K.; Forsberg, M.; Zwahlen, M.; Kampf, C.; Wester, K.; Hober, S.; et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 2010, 28, 1248–1250. [CrossRef] 23. Thul, P.J.; Åkesson, L.; Wiking, M.; Mahdessian, D.; Geladaki, A.; Blal, H.A.; Alm, T.; Asplund, A.; Björk, L.; Breckels, L.M.; et al. A subcellular map of the human proteome. Science 2017, 356, eaal3321. [CrossRef] [PubMed] 24. Brooks, B.R.; Miller, R.G.; Swash, M.; Munsat, T.L. El Escorial revisited: Revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Mot. Neuron. Disord. 2000, 1, 293–299. [CrossRef] 25. Williams, S.M.; Khan, G.; Harris, B.T.; Ravits, J.; Sierks, M.R. TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis. BMC Neurosci. 2017, 18, 1–12. [CrossRef] 26. Codron, P.; Cassereau, J.; Vourc’H, P.; Veyrat-Durebex, C.; Blasco, H.; Kane, S.; Procaccio, V.; Letournel, F.; Verny, C.; Lenaers, G.; et al. Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions. Amyotroph. Lateral Scler. Front. Degener. 2018, 19, 446–456. [CrossRef] 27. Ren, Y.; Liu, W.; Li, Y.; Sun, B.; Li, Y.; Yang, F.; Wang, H.; Li, M.; Cui, F.; Huang, X. Cutaneous somatic and autonomic nerve TDP-43 deposition in amyotrophic lateral sclerosis. J. Neurol. 2018, 265, 1753–1763. [CrossRef] 28. Ono, S. The skin in amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Mot. Neuron Disord. 2000, 1, 191–199. [CrossRef] 29. Zhang, H.; Tan, C.-F.; Mori, F.; Tanji, K.; Kakita, A.; Takahashi, H.; Wakabayashi, K. TDP-43-immunoreactive neuronal and glial inclusions in the neostriatum in amyotrophic lateral sclerosis with and without dementia. Acta Neuropathol. 2007, 115, 115–122. [CrossRef] 30. Paré, B.; Gros-Louis, F. Potential skin involvement in ALS: Revisiting Charcot’s observation–a review of skin abnormalities in ALS. Rev. Neurosci. 2017, 28, 551–572. [CrossRef] [PubMed] 31. Neumann, M.; Kwong, L.K.; Lee, E.B.; Kremmer, E.; Flatley, A.; Xu, Y.; Lee, V.M.Y. Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies. Acta Neuropathol. 2009, 117, 137–149. [CrossRef] [PubMed] 32. Hasegawa, M.; Arai, T.; Nonaka, T.; Kametani, F.; Yoshida, M.; Hashizume, Y.; Beach, T.G.; Buratti, E.; Baralle, F.; Morita, M.; et al. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Ann. Neurol. 2008, 64, 60–70. [CrossRef] 33. Miki, Y.; Mori, F.; Nunomura, J.; Ookawa, K.; Yajima, N.; Yagihashi, S.; Wakabayashi, K. Sporadic amyotrophic lateral sclerosis with pallido-nigro-luysian degeneration: A TDP-43 immunohistochemical study. Neuropathology 2010, 30, 149–153. [CrossRef] 34. Igaz, L.M.; Kwong, L.K.; Xu, Y.; Truax, A.C.; Uryu, K.; Neumann, M.; Clark, C.M.; Elman, L.B.; Miller, B.L.; Grossman, M.; et al. Enrichment of C-Terminal Fragments in TAR DNA-Binding Protein-43 Cytoplasmic Inclusions in Brain but not in Spinal Cord of Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Am. J. Pathol. 2008, 173, 182–194. [CrossRef] 35. Nishihira, Y.; Tan, C.-F.; Onodera, O.; Toyoshima, Y.; Yamada, M.; Morita, T.; Nishizawa, M.; Kakita, A.; Takahashi, H. Sporadic amyotrophic lateral sclerosis: Two pathological patterns shown by analysis of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions. Acta Neuropathol. 2008, 116, 169–182. [CrossRef] 36. Weskamp, K.; Tank, E.M.; Miguez, R.; McBride, J.P.; Gómez, N.B.; White, M.; Lin, Z.; Gonzalez, C.M.; Serio, A.; Sreedharan, J.; et al. Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS. J. Clin. Investig. 2020, 130, 1139–1155. [CrossRef] 37. Weis, J.; Katona, I.; Muller-Newen, G.; Sommer, C.; Necula, G.; Hendrich, C.; Ludolph, A.C.; Sperfeld, A.-D. Small-fiber neuropathy in patients with ALS. Neurology 2011, 76, 2024–2029. [CrossRef] [PubMed] 38. de Carvalho, M.; Dengler, R.; Eisen, A.; England, J.D.; Kaji, R.; Kimura, J.; Swash, M. Electrodiagnostic criteria for diagnosis of ALS. Clin. Neurophysiol. 2008, 119, 497–503. [CrossRef] 39. Shefner, J.M.; Al-Chalabi, A.; Baker, M.R.; Cui, L.-Y.; de Carvalho, M.; Eisen, A.; Grosskreutz, J.; Hardiman, O.; Henderson, R.; Matamala, J.M.; et al. A proposal for new diagnostic criteria for ALS. Clin. Neurophysiol. 2020, 131, 1975–1978. [CrossRef] 40. Palese, F.; Sartori, A.; Logroscino, G.; Pisa, F.E. Predictors of diagnostic delay in amyotrophic lateral sclerosis: A cohort study based on administrative and electronic medical records data. Amyotroph. Lateral Scler. Front. Degener. 2019, 20, 176–185. [CrossRef] [PubMed] 41. Paganoni, S.; Macklin, E.; Lee, A.; Murphy, A.; Chang, J.; Zipf, A.; Cudkowicz, M.; Atassi, N. Diagnostic timelines and delays in diagnosing amyotrophic lateral sclerosis (ALS). Amyotroph. Lateral Scler. Front. Degener. 2014, 15, 453–456. [CrossRef] 42. Feneberg, E.; Gray, E.; Ansorge, O.; Talbot, K.; Turner, M. Towards a TDP-43-Based Biomarker for ALS and FTLD. Mol. Neurobiol. 2018, 55, 7789–7801. [CrossRef] 43. Verstraete, E.; Kuiperij, H.B.; Van Blitterswijk, M.M.; Veldink, J.H.; Schelhaas, H.J.; Van Den Berg, L.H.; Verbeek, M.M. TDP-43 plasma levels are higher in amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. 2012, 13, 446–451. [CrossRef] Cells 2022, 11, 209 12 of 12 44. De Marco, G.; Lomartire, A.; Calvo, A.; Risso, A.; De Luca, E.; Mostert, M.; Chiò, A. Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution. Neuropathol. Appl. Neurobiol. 2017, 43, 133–153. [CrossRef] 45. De Marco, G.; Lupino, E.; Calvo, A.; Moglia, C.; Buccinna, B.; Grifoni, S.; Chio, A. Cytoplasmic accumulation of TDP-43 in circulating lymphomonocytes of ALS patients with and without TARDBP mutations. Acta Neuropathol. 2011, 121, 611–622. [CrossRef] [PubMed] 46. Junttila, A.; Kuvaja, M.; Hartikainen, P.; Siloaho, M.; Helisalmi, S.; Moilanen, V.; Kiviharju, A.; Jansson, L.; Tienari, P.J.; Remes, A.M.; et al. Cerebrospinal Fluid TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis Patients with and without the C9ORF72 Hexanucleotide Expansion. Dement. Geriatr. Cogn. Disord. Extra 2016, 6, 142–149. [CrossRef] [PubMed] 47. Noto, Y.-I.; Shibuya, K.; Sato, Y.; Kanai, K.; Misawa, S.; Sawai, S.; Mori, M.; Uchiyama, T.; Isose, S.; Nasu, S.; et al. Elevated CSF TDP-43 levels in amyotrophic lateral sclerosis: Specificity, sensitivity, and a possible prognostic value. Amyotroph. Lateral Scler. 2010, 12, 140–143. [CrossRef] 48. Kasai, T.; Tokuda, T.; Ishigami, N.; Sasayama, H.; Foulds, P.; Mitchell, D.J.; Mann, D.M.A.; Allsop, D.; Nakagawa, M. Increased TDP-43 protein in cerebrospinal fluid of patients with amyotrophic lateral sclerosis. Acta Neuropathol. 2009, 117, 55–62. [CrossRef] 49. Steinacker, P.; Hendrich, C.; Sperfeld, A.D.; Jesse, S.; von Arnim, C.A.F.; Lehnert, S.; Pabst, A.; Uttner, I.; Tumani, H.; Lee, V.M.-Y.; et al. TDP-43 in Cerebrospinal Fluid of Patients with Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Arch. Neurol. 2008, 65, 1481–1487. [CrossRef] [PubMed] 50. Olsson, B.; Lautner, R.; Andreasson, U.; Öhrfelt, A.; Portelius, E.; Bjerke, M.; Hölttä, M.; Rosén, C.; Olsson, C.; Strobel, G.; et al. CSF and blood biomarkers for the diagnosis of Alzheimer’s disease: A systematic review and meta-analysis. Lancet Neurol. 2016, 15, 673–684. [CrossRef]