Fluid biomarkers unveil signatures of pathological aging

Aging is a multifaceted and highly varied process in the brain. Identifying aging biomarkers is one means of distinguishing pathological from physiological aging. The aim of this narrative review is to focus on two new developments in the field of fluid biomarkers and draw attention to this excellent tool for the early detection of potential brain pathologies that delay, alter, or enable physiological aging to become pathological. Pathological aging can lower the threshold for the development of specific diseases such as late-onset epilepsy. Fluid biomarkers can reveal pathological levels at an early stage and thus indicate disease processes in the brain that begin before symptoms develop; they thus differ from physiological aging.


Aging biomarkers
Aging is caused by various processes in the brain and takes place in different regions of the brain.Recent concepts include inflammaging (Komleva et al., 2021;Giunta et al., 2008), which focus on inflammatory pathways activated to varying degrees as we age.Neurodegeneration also occurs in physiological aging, but differs considerably from pathological aging in its extent.
Healthy aging is sometimes difficult to differentiate from pathological aging in conditions revealing symptoms of cognitive decline.One reason for this may be that symptoms can be subjective and sometimes fail to appear during neuropsychological testing procedures, for example.Thorough diagnosis is therefore necessary to determine whether cognitive decline's initial, subjective symptoms can already be attributed to a brain pathology.Although the number of imaging techniques to visualize individual pathologies in aging individuals keeps growing, they are often specific, such as amyloid positron emission tomography for pathological amyloid beta deposits in the brain in Alzheimer´s disease (AD) or single photon emission computed tomography involving ioflupane I123 injections to visualize dopamine transporter density for detecting alpha-synucleinopathies, which only yields indications of certain pathological processes.In contrast, the examination of blood or cerebrospinal fluid as fluid biomarkers allows the simultaneous investigation of different brain pathologies.Fluid biomarkers can therefore be regarded as an indispensable tool for the simultaneous visualization of different biological processes in the brain.Blood tests are less specifically suited for visualizing brain processes, but blood biomarkers have been gaining new importance and appreciation in  However, we still need more standardization of blood-based multi-parameters in dementia diagnostics.Here, automated and already validated assays for biofluid marker based dementia diagnostics in the CSF are the most promising.Furthermore, in real-life diagnostic scenarios, a single blood biomarker such as p-tau217 or Aβ1-42/1-40 cannot meet these intricate differential diagnostic requirements -in particular at preclinical stages such as subjective cognitive decline due to AD. Therefore there is an urgent requirement for research to identify the most valid and reliable multi-parameter signature utilizing different fluid biomarkers -preferentially using automated platforms for staging pathological aging.Overall, it is now clear that AD's pathological processes start early and therefore differ from physiological aging and characterize pathological aging.However, it is also important to note that neuronal cell damage in the form of elevated tau protein but also amyloidopathy can also occur in acquired, non-lesional late- For this form of pathological aging, it is important to assess neurodegeneration markers, especially amyloid beta peptides (Table 1, Figure 1).However, the determination of biomarkers is not only potentially relevant in the context of a possible preclinical AD: recently published study results indicate that amyloid beta and tau protein markers in histological preparations from epilepsy surgery were even elevated in patients with medial temporal lobe epilepsy attributable to limbic encephalitis (Rácz et al., 2022).It is therefore clear that neurodegeneration markers can also develop in connection with pathological aging characterized by LOEU or as a result of chronic autoimmune inflammation.

Fluid biomarkers as a tool for establishing signatures of autoimmunity
Inflammatory and immune processes in the brain are other very important factors that change with age.Immune ageing is also regarded as an aging process that includes pathological alterations within the immune system.Various processes are found in the immune system during the process of aging like degeneration of the thymus, spleen and lymph nodes, as well as a reduction in the frequency of naïve B cells and the capacity to produce antibodies (Aging Biomarker Consortium, 2023) or alterations in macrophages (Mogilenko et al., 2022).There is also evidence of an altered B-cell subpopulation in old age, which has an influence on Toll-like receptor 7 and Toll-like receptor 9 signaling in conjunction with Th1 cytokines (Cancro, 2020).
It is assumed that this B-cell subpopulation contributes to the reduced B-cell production in old age and a weaker immune response (Cancro, 2020) Ultimately, as the pathogenicity of neural autoantibodies associated with cognitive impairment has not been conclusively clarified, the question arises as to whether such autoantibodies are also increasingly detectable in healthy people -as suggested by a larger study involving over 7000 patients and 49 tested antibodies (Daguano Gastaldi et al., 2022) and thus also potentially co-existing with healthy ageing.However, there is ample evidence indicating that specific autoantibodies play a potentially pathogenic role, such as KCNA2 autoantibodies.The study (Arlt et al., 2024) reported detecting immune cells in the tissue and KCNA2 antibodies bound to the hippocampus and cerebellum of animal slices.There is thus indirect evidence of these antibodies' pathogenicity.Other transfer models from humans to animals have shown that other antibodies against glutamatergic receptors can cause synaptic transmission and thus cognitive disorders (Haselmann et al., 2018).Autoantibodies occurring in old age could therefore be pathogenic.Further research is required to assess the significance of neural autoantibodies specifically in conjunction with pathological aging, but also their occurrence and significance in healthy aging.

Novel proteomic and genomic approaches for investigating pathological aging
The analysis of fluid biomarkers is important when assessing neurodegenerative and autoimmune processes in pathological aging.Fluid biomarkers have been analyzed by taking hypothesis-driven approaches.However, it is important to consider unbiased genomic and proteomic interactions to acquire deeper understanding of neurodegenerative processes and immune-inflammatory processes in pathological aging.Plasma and brain tissue, and a genomic atlas of different protein levels were generated in a recent CSF analysis (Yang et al., 2021).
Important proteins associated with AD were identified applying the randomized mendelization method (Yang et al., 2021).Such tissue examinations are therefore very valuable to correlate results from genome-wide association studies (GWAS) studies with functionally relevant genes.
They also play an important role in immune-inflammatory autoantibody-associated diseases, ie, neurological syndromes with glutamic acid decarboxylase 65 (GAD65) positivity via genome-wide testing, and a human leukocyte antigen system analysis in a large cohort of 1214 participants (including 167 patients with neurological syndrome and GAD65 autoantibodies) (Strippel et al., 2024).The GWAS findings were validated on a CSF proteome and a total of 16 relevant gene loci in a neurological syndrome with GAD65 autoantibodies identified (Strippel et al., Another proteomic CSF examination revealed a dysregulated complement system in patients with autoimmune encephalitis compared to controls, as well as additional deficits in immune regulation and the nerve-cell function (Räuber et al., 2023), making it clear that proteomic and genomic analyses of brain tissue, but also of fluid biomaterial, should accompany the biomarker analyses mentioned above.

Immune cell signatures in pathological aging
To confirm the relationship between clinical features and relevant immune cell profiles in the CSF and blood, a multidimensional examination of immune cells in blood and CSF could prove to be groundbreaking, as has been shown prototypically for neurological diseases in the study by Gross et al. (Gross et al. 2021).In their study, the features were selected by reducing the dimensionality in combination with machine learning methods (Gross et al., 2021).The disease parameters in our study mainly involved immune cells.Such an approach may help to generate subtypes of CNS-driven autoimmunity in pathological aging.By using multidimensional flow cytometry in the CSF together with investigations of immune subset cell populations, it is possible to distinguish psychotic diseases from inflammatory diseases (Räuber et al., 2021).
Such an approach could also help when assessing the pathological aging process to distinguish physiological aging from pathological aging involving organically caused diseases.

Synopsis and perspective
By examining fluid biomarkers, we may eventually be able to help clarify the brain's transition from healthy to diseased.Neurodegenerative processes as pathological aging of the brain can also cause increased epileptic discharges, as recently demonstrated in Lewy body dementia patients (Musaeus et al., 2023).In a larger study of 1251 patients, it was exhibited that more cortical thinning as well as white matter structural abnormalities were existent on MRI in patients with late-onset epilepsy prior to the onset of seizures (  (Romoli et al., 2021).Fluid biomarkers represent an excellent opportunity to detect processes of pathological ageing some 10-20 years before the onset of clinical symptoms.
However, such early diagnosis also raises new ethical issues that warrant discussion.The potential value of unmasking pathological ageing processes has risen considerably.However, this often fails to clarify whether a pathological process is already present, because this is about more than just detecting a pathology, namely, how we define a disease such as epilepsy and AD and harmonize this with the commitment to treating certain symptoms.

18 and 9
years prior to diagnosis.The discovery of blood biomarkers for AD could enable decisive advances in our understanding of ageing within just a few years.Such biomarker anomalies before the onset of sporadic AD symptoms were confirmed in another study: the Aβ ratio 42/40 in CSF and plasma and p-tau217 in CSF changed 15-19 years before symptom onset(Li et al. 2024).Pathological changes in plasma levels of 1) p-tau217, glial fibrillary acidic protein (GFAP) and Nfl in plasma, as well as 2) a synaptosomal-associated protein of 25 kDa (SNAP25), neurogranin and myeloid cell expressed receptor 2 (TREM2) in the CSF were detected 12-14 years before the onset of cognitive-impairment symptoms(Li et al., 2024).Within a 10-year period before symptom diagnosis, p-tau205 and YKL-40 biomarkers in the CSF were already revealing pathological abnormalities(Li et al., 2024).What this evidence indicates is that AD's neurodegenerative trajectory is already existent up to 20 years prior to the onset of symptoms.The diagnosis of ageing is gaining whole new meaning, especially in the case of preliminary stages of dementia including mild cognitive impairment, but it is even generating novel ethical challenges.The measurement of the epitopes of beta-amyloid peptides and phosphorylated tau protein in blood probes will soon be part of routine neurochemical dementia diagnostics due to its high diagnostic accuracy, minimal invasiveness and relative insensitivity.There is evidence that p-tau217 is already elevated in preclinical and prodromal AD(Saloner et  al., 2024; Ashton et al., 2024) -before phospho-tau181 becomes elevated in blood and the CSF -and that it can predict cognitive decline (Mattson Carlgren et al., 2023).

Figure 1 :
Fluid biomarkers track pathological ageingFluid biomarkers are crucial to distinguishing pathological from physiological aging.Pathological aging might lead to late-onset epilepsy.
conjunction with certain diseases of old age such as AD (Perneczyk et al., 2024; Morgado et al., 2024, Klafki et al., 2022; Teunissen et al., 2023; Therriault et al., 2024; Nakamura et al., 2018; Barthélemy et al., 2024).The aim of this review is to briefly describe the most recent developments in fluid and particular blood biomarkers in conjunction with two exemplary pathologies of the brain occurring in old age, such as AD pathology, but also inflammatory pathologies in autoantibody-associated diseases in old age.We provide an insight into their current scientific significance and describe in this narrative review how such pathologies as signatures of neurodegeneration and autoimmunity can be detected in aging processes.This focused review is narrative only, and not a systemic review.In brief, we conducted a PubMed search applying the following terms: fluid biomarkers, blood, CSF, neurodegeneration, inflammaging, autoantibodies, pathological aging, AD, autoimmune encephalitis, late onset epilepsy in combination or alone.Articles were chosen for this review at the authors subjective discretion. 2 Fluid biomarkers as signatures of neurodegeneration AD is a prime example of pathological aging.Although its diagnostic criteria now biologically rely on biomarkers, there is also a clinical entity (Dubois et al., 2021).The prominent clinical phenotype of AD is hippocampus dependent memory impairment, which certainly overlaps with mnestic syndromes as seizure disorders such as temporal lobe epilepsy or limbic encephalitis.There are also other rare phenotypes of AD such as posterior cortical atrophy as well as the logopenic variant of primary progressive aphasia (Dubois et al., 2021).There have been suggestions to consider AD as a biological-clinical entity (Dubois et al. - Zheng et al., 2023)igger an altered immune defense and thus also affect autoimmunity processes in old age.However, age- Zheng et al., 2023).T-cell-dependent aging is already considered a risk factor for the development of autoimmunity(Liu et al., 2022).There is additional evidence that epigenetic mechanisms also contribute to immunosenescence and the development of after initial immunotherapy in the first year, which would benefit from renewed immunotherapy(Muñoz-Lopetegi et al., 2023).This circumstance emphasizes the importance of targeted diagnostics of pathological processes in old age.
(Aboseif et al., 2023) very effective at producing autoantibodies.However, a certain predisposition is required for immune aging involving alterations in T-cell aging leading to a chronic inflammatory state also referred to as inflammaging, leading to autoimmune inflammation (autoantibodies against its own antigens is not affected.There are many new autoantibodies to intracellular and membrane surface antigens not yet reported to coincide with cognitive impairment(Hansen, 2021).Neural autoantibodies associated with cognitive disorders in old age have been identified, such as flotillin 1/ 2, recoverin or potassium voltage-gated channel subfamily A member 2 (KCNA2), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR), IgLON5, Leucin Rich Glioma Inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2) (Hansen et al., 2021, Timäus et al., 2021; Arlt et al., 2024; Hansen et al., 2022; Blinder and Lewerenz, 2019).Nevertheless, their significance is poorly understood, and large cohort studies are still needed.It is important to better understand normal aging's phenomenology in order to recognize subtle changes and investigate these via blood biomarkers antibodies(Arlt et al., 2024).In addition, there are other neural autoantibodies such as LGI1, which lead to cognitive impairment in old age, but also to seizures(Aboseif et al., 2023).In addition, a case series has shown that LGI1 patients often continue to have videopolysomnographic abnormalities even (Sarkis et al., 2019)There is thus evidence that pathological aging in form of neurodegenerative processes can favor the occurrence of late-onset epilepsy.The 20-50% of all epilepsies occurring in old age without a clear etiology are attributable to LOEU(Sarkis et al., 2019), whereas the rest of late onset epilepsy tends to be lesional.A recent study of 54 people with non-lesional LOEU found that the main seizure type was characterized by bilateral tonic-clonic seizure (30%), by motor