Cutaneous pTDP-43 Deposits Distinguish ALS From Other Neuropathies

Peripheral Protein Pathology in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative condition traditionally defined by motor neuron loss, yet emerging evidence characterizes it as a multisystem disorder involving sensory pathways [1]. Sensory neuropathy, affecting both large and small fibers, may be present in up to 20% of patients and often complicates the clinical presentation [1]. While diagnostic protocols for other protein-misfolding disorders, such as transthyretin amyloidosis, rely heavily on tissue biopsies to identify pathogenic deposits, the diagnosis of amyotrophic lateral sclerosis remains primarily clinical [2]. The lack of a definitive peripheral biomarker contributes to diagnostic delays, which can postpone critical interventions such as non-invasive ventilation that are known to improve survival outcomes [3]. Identifying objective indicators of disease pathology is essential for accurate staging and monitoring the accumulation of cellular damage over time [4]. To address this diagnostic gap, researchers recently investigated whether peripheral tissue analysis, specifically skin and tongue biopsies, could provide a reliable premortem biomarker for the disease.

Quantifying Cutaneous Protein Accumulation

The cross-sectional case-control study evaluated 50 patients with amyotrophic lateral sclerosis who met the revised El Escorial criteria, a standardized clinical framework used to diagnose the disease. This patient cohort had a median age of 66.5 years and was 36% female. To establish a rigorous comparison, the researchers recruited two distinct control groups: 20 healthy individuals with a median age of 60 years (20% female) and 20 patients with non-ALS neuropathy or neuronopathy with a median age of 60 years (50% female). By including this latter group, the authors aimed to determine whether any observed protein pathology was specific to amyotrophic lateral sclerosis or merely a general feature of peripheral nerve damage.

The researchers focused on phosphorylated TAR DNA-binding protein 43 (pTDP-43), the central pathologic hallmark of amyotrophic lateral sclerosis. Using standardized immunofluorescence (a laboratory technique that uses fluorescent antibodies to visualize specific proteins in tissue), they quantified pTDP-43 within keratinocytes and Meissner corpuscles, the specialized mechanoreceptors in the skin responsible for detecting light touch. Within these Meissner corpuscles, the team assessed the percentage of Protein Gene Product 9.5 (PGP, a pan-neuronal marker), the percentage of pTDP-43, and the pTDP-43/PGP ratio. This methodology allowed for a precise measurement of pathological protein accumulation relative to the remaining nerve fiber density in the skin.

The findings demonstrated that pTDP-43 deposits were present across epidermis and dermis structures in patients with amyotrophic lateral sclerosis, whereas they were almost absent in healthy controls and found at only low levels in the non-ALS neuropathy group. Specifically, the percentage of pTDP-43 in Meissner corpuscles differed significantly across the three groups (H = 53.30; p < 0.001). The median pTDP-43 percentage in Meissner corpuscles was 0.35 (interquartile range 0.39) in the amyotrophic lateral sclerosis group, compared to 0.04 (interquartile range 0.03) in the non-ALS neuropathy group and 0.00 in healthy controls. For clinicians, these data indicate that the accumulation of this specific phosphorylated protein in the skin is highly characteristic of amyotrophic lateral sclerosis, raising the possibility of using minimally invasive skin biopsies to secure a definitive tissue diagnosis.

Correlation With Clinical Disease Progression

To determine if the density of peripheral protein deposits reflects the clinical advancement of the disease, the researchers evaluated the relationship between biopsy findings and functional impairment. In this study, disease severity was classified using the King's staging system, a clinical framework for tracking progression from initial symptom onset through to nutritional or respiratory failure. By aligning the tissue data with these clinical stages, the authors sought to establish whether cutaneous protein accumulation could serve as a reliable proxy for central nervous system disease burden.

The analysis focused on the pTDP-43/PGP ratio, which measures the amount of pathological protein relative to the remaining healthy neuronal markers in the skin. The researchers found that the pTDP-43/PGP ratio increased with clinical stage at the subject level (Z = 2.20, p = 0.028). This correlation suggests that as patients advance through the clinical stages of the disease, the accumulation of phosphorylated protein in the skin becomes more pronounced, directly mirroring the worsening clinical picture.

This association between protein accumulation and disease severity was further confirmed at a more granular level of analysis. The pTDP-43/PGP ratio also increased with clinical stage at the single-corpuscle level (H = 21.72, p < 0.001), indicating that individual sensory receptors accumulate higher concentrations of the pathological protein as the disease progresses. For the practicing clinician, these findings suggest that peripheral pathology is a dynamic reflection of the systemic disease process. This supports the potential use of serial skin biopsies not just for initial diagnosis, but as an objective biomarker to monitor disease severity and progression over time.

Diagnostic Accuracy and Tissue Specificity

To confirm the initial findings, the researchers performed a western blot analysis, a laboratory technique used to separate and quantify specific proteins in a tissue sample. This analysis showed a higher pTDP-43/GAPDH ratio in the skin of patients with amyotrophic lateral sclerosis than in healthy controls, with median values of 3.45 (interquartile range 7.23) compared to 1.30 (interquartile range 0.80; p = 0.007). Crucially, the levels of nonphosphorylated TDP-43 did not differ across the study groups. This distinction is clinically significant because it indicates that the pathology is specific to the phosphorylated (chemically modified) form of the protein rather than a general overabundance of the native protein, which remains stable across healthy and diseased states.

The diagnostic utility of these findings was further supported by the analysis of keratinocytes, the primary cell type in the epidermis. In these cells, pTDP-43 levels were significantly higher in patients with amyotrophic lateral sclerosis (median 0.047, interquartile range 0.023) than in those with non-ALS neuropathy or neuronopathy (median 0.019, interquartile range 0.049) and healthy controls (median 0.005, interquartile range 0.047; p < 0.001). When the researchers combined various cutaneous pTDP-43 measures, the diagnostic accuracy was exceptionally high. The combined measures discriminated amyotrophic lateral sclerosis from healthy controls with an area under the curve of 0.94 (p < 0.001) and from non-ALS neuropathy or neuronopathy with an area under the curve of 0.90 (p < 0.001). For clinicians, an area under the curve (a statistical measure of diagnostic accuracy where 1.0 represents a perfect test) of 0.90 or higher suggests that skin biopsy could serve as a highly reliable tool for differentiating this fatal disease from other mimicking neurological conditions.

Beyond the skin, the study investigated the presence of pathology in the tongue, a site frequently involved in bulbar-onset disease. Tongue biopsies were performed in 10 patients with amyotrophic lateral sclerosis and 10 patients with burning mouth syndrome, a condition characterized by chronic oral pain without an obvious cause. In the amyotrophic lateral sclerosis group, tongue biopsies revealed pTDP-43 aggregates in intramuscular nerves, denervated endplates, and muscle fibers. These findings demonstrate that the pathological protein is not limited to the skin but is also present in the specialized tissues of the tongue. Ultimately, this reflects the widespread nature of the proteinopathy and suggests that peripheral tissue biopsies could soon provide physicians with a definitive, premortem diagnostic tool.

References

1. Bombaci A, Lupica A, Pozzi FE, Remoli G, Manera U, Stefano VD. Sensory neuropathy in amyotrophic lateral sclerosis: a systematic review.. Journal of neurology. 2023. doi:10.1007/s00415-023-11954-1

2. Adams D, Ando Y, Beirão JM, et al. Expert consensus recommendations to improve diagnosis of ATTR amyloidosis with polyneuropathy. Journal of Neurology. 2020. doi:10.1007/s00415-019-09688-0

3. Ward SLD. Randomised controlled trial of non-invasive ventilation (NIV) for nocturnal hypoventilation in neuromuscular and chest wall disease patients with daytime normocapnia. Thorax. 2005. doi:10.1136/thx.2004.037424

4. Liguori I, Russo G, Curcio F, et al. Oxidative stress, aging, and diseases. Clinical Interventions in Aging. 2018. doi:10.2147/cia.s158513