Phosphoproteomics-guided tau biomarker discovery in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD)

suggests that tau phosphorylation is not only an AD signature but may represent a broader pathological axis across multiple NDDs [6]. The advent of phosphoproteomics has transformed our ability to map disease-relevant phosphorylation sites with unprecedented resolution. By illuminating aberrant signaling cascades, phosphoproteomics provides powerful opportunities for biomarker discovery and therapeutic targeting [7]. Herein, in this opinion, we discuss how phosphoproteomicsguided analysis of tau phosphorylation is redefining biomarker strategies in both AD and ALS, bridging two diseases once considered molecularly distinct.Phosphorylated tau species, particularly p-tau217 and p-tau 181, have emerged as robust diagnostic biomarkers in Alzheimer's disease (AD) [8]. Importantly, these phosphorylated tau variants were identified in muscle biopsies from ALS patients and localized to atrophic fibers using immunohistochemistry and mass spectrometry techniques. This peripheral source of p-tau challenges the CNS-centric perspective of tau pathology and raises intriguing questions about common phosphorylation mechanisms in both the central and peripheral nervous systems [5].Phosphorylated tau has recently been identified as a unifying biomarker axis in AD and ALS, two conditions previously regarded as molecularly distinct. In AD, plasma p-tau 217 and p-tau 181 exhibit superior diagnostic accuracy relative to other biomarkers, with ptau 217 increasing early in the disease and demonstrating a robust correlation with amyloid deposition [5,9]. Furthermore, incorporating plasma neurofilament light chain (NfL) ratios, a marker of neuroaxonal damage, enhances the test's specificity, as demonstrated in large clinical groups, thereby reducing the need for invasive CSF testing [10]. Interestingly, concurrent advancements in ALS suggest that increased serum levels of p-tau 217 and p-tau 181 are associated with enhanced motor neuron dysfunction, increased clinical severity, and lower motor neuron (LMN)-dominant phenotypes. Even more surprisingly, phosphorylated tau species have been found in muscle biopsies from ALS patients [5]. This suggests that tau pathology can originate from outside the CNS. These findings collectively contest the conventional distinctions between AD and ALS, indicating a common framework for phosphoproteomics. P-tau 217 is an emerging blood-based biomarker; specifically, it stands out as a promising crossdisease biomarker, serving as an early indicator of amyloid-related pathology in AD.However, it reflects LMN injury and kinase dysregulation in ALS [5]. Combining tau phosphorylation with markers like NfL could lead to multiplexed biomarker panels that can distinguish subtypes, differentiate between overlapping phenotypes, and inform cross-disease treatment plans Figure 1.. Earlier studies have indicated that p-tau181 levels increase due to LMN involvement and degeneration, leading to higher p-tau181 in the plasma of ALS patients. However, some reports suggest that p-tau181 and p-tau217 may be released from peripheral axons and nearby denervated muscle fibers [16]. Additionally, pathological tau species have been detected in the blood and other peripheral tissues, such as skin and nerves, in patients with progressive supranuclear palsy, a tauopathy, which supports the presence of tau outside the CNS [17]. This cumulative evidence suggests the peripheral involvement of tau release from the denervated muscle fiber and thus the peripheral involvement in the neurological conditions. However, further studies are warranted with a large number of cohorts, including patients from each stage of clinical progression in ALS because of the heterogeneous nature of the disease.Both plasma p-tau181 [18] and p-tau181 [19] levels are reported to be increased in the early stages of AD or preclinical stages of AD. Both begin to rise in individuals who are cognitively normal but have early AD (defined by amyloid-beta positivity). Levels of p-tau181 and p-tau217 are higher in people with mild cognitive impairment and continue to increase over time, correlating with a decline in cognition and brain atrophy. The significant milestone is the recent approval of the development Elecsys pTau181 biomarker test, developed by two pharmaceutical companies, Roche in Basel, Switzerland, and Eli Lilly in Indianapolis, for the detection of AD [20].In case of ALS neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) is elevated in presymptomatically and continue to rise in the early stages of the disease and recently regarded as promising prognostic and diagnostic biomarkers [21],nevertheless, no validated studies have shown the consistency of increase in p-tau181 and p-tau217 during the clinical progression of ALS starting from presymptomatic phase to symptomatic phase. The landmark study by Rumeileh and his team has caused a paradigm shift in how we think about peripheral involvement of taupathy in ALS.Additionally, patient stratification studies that combine neurofilament involvement stages with peripheral tau pathology might help solve the challenge of distinguishing between AD and ALS.Phosphoproteomics is transforming NDDs research by identifying both p-tau181 and p-tau217 as molecular links between AD and ALS. This connection opens the door for cross-disease biomarker strategies and the development of personalized therapies. Tau phospho-biology, therefore, serves as a unifying focus to address the complexity of currently untreatable NDDs. However, recent studies have prompted a paradigm shift in how we think about the involvement of peripheral tissues in NDDs. To tackle the challenges of overlapping diagnoses among different NDDs, we should consider the varied involvement of CNS and peripheral tissues at each stage of clinical progression, assessed longitudinally. Additionally, future large cohort studies with ALS and AD muscle biopsies are necessary to verify the findings of increased p-tau181 and p-tau217 using the reliable, reproducible, robust, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with data-independent acquisition (DIA) phosphoproteomics techniques.