Serum Cathepsin S Levels Do Not Show Alterations in Different Clinical,Neuropathological,or Genetic Subtypes of Frontotemporal Dementia Patients nor in Comparison to Healthy Control Individuals

INTRODUCTION

Frontotemporal dementia (FTD) is an umbrella term for a group of clinical syndromes characterized by atrophy in frontal and temporal regions. These syndromes include behavioral variant frontotemporal dementia (bvFTD), primary progressive aphasias, the FTD-plus phenotypes, namely corticobasal syndrome and progressive supranuclear palsy (PSP), and FTD with concomitant motoneuron disease (FTD-MND) [1, 2]. The definite diagnosis of FTD remains a challenge, especially in the early phases of disease, due to the overlapping cognitive, motor, and neuropsychiatric features with other neurodegenerative diseases and primary psychiatric disorders as well as due to the lack of disease-specific biomarkers. Early accurate diagnosis of FTD is a crucial step toward developing disease-modifying therapies and effective disease management strategies. Two major neuropathological subtypes, the TAR DNA-binding protein 43 (TDP-43) proteinopathies and tauopathies, account for approximately 90 % of FTD cases [2]. The C9orf72 hexanucleotide repeat expansion (C9-HRE) and GRN mutations associate with TDP-43 proteinopathy in FTD patients, whereas FTD patients carrying mutations in the microtubule-associated protein tau gene (MAPT) are characterized by tau-positive inclusions. Interestingly, specific mutations in GRN, gene encoding progranulin protein, have also been shown to link with lysosomal storage disease in addition to FTD spectrum diseases [3].

Lysosomes are cellular organelles involved in the degradation and recycling of intracellular and extracellular material, including lipids, proteins, nucleic acids, and carbohydrates [4]. Autophagy, on the other hand, is a process responsible for the sequestration and degradation of long-lived proteins, dysfunctional organelles, and invading pathogens. This degradation process takes place in autolysosomes, which form upon the fusion of autophagosomes with lysosomes [5]. Recent studies have provided evidence that lysosomal dysfunction associates with the onset and progression of FTD and amyotrophic lateral sclerosis (ALS) [6]. Mutations in several genes encoding proteins that are mechanistically related to lysosomal and autophagosomal function, including C9-HRE, sequestome 1 (SQSTM1/p62), ubiquilin 2 (UBQLN2), dynactin subunit 1 (DCTN1), TANK-binding kinase 1 (TBK1), optineurin (OPTN), and valosin containing protein (VCP) have been linked to both diseases [7]. Interestingly, many of these genes are highly expressed in microglia, which are key immune cells of the brain. These proteins localize to lysosomes or modulate their function, trafficking, lysosomal acidification, and autophagy.

The most abundant lysosomal proteases are the family of cathepsins that play a vital role in protein degradation, energy metabolism, and immune responses [8]. There are eleven cathepsins encoded in the human genome (B, C, F, H, K, L, O, S, V, W, and X) [9]. Cathepsin B is the most studied cathepsin expressed in the central nervous system (CNS), and its elevated levels in serum correlate with cognitive dysfunction in Alzheimer’s disease (AD) patients [10]. Cathepsins have also been shown to cleave progranulin [11], which regulates inflammatory responses in the CNS and is linked to FTD pathophysiology [12]. Cysteine cathepsins, such as cathepsin S, have been reported to contribute to neurodegeneration and deposition of protein aggregates in neuronal cells [13] and associate with AD progression [14]. Cathepsin S is expressed throughout in the brain and body, but is preferentially expressed in the microglia in the CNS, suggesting that it impacts their function and interaction with neurons [15]. Cathepsin S is unique among the cysteine cathepsin family due to its restricted tissue expression, association with antigen-presenting cells localized in lymph nodes and spleen as well as other immune cells, such as macrophages and microglia, and its ability to remain active in environments with neutral pH, such as extracellular space [16]. Therefore, cathepsins, and especially cathepsin S, might be interesting biomarker candidates in FTD, but has remained to be studied further.

Here, we have used the ultrasensitive single molecule array (Simoa) method to measure the levels of serum cathepsin S in a large multicenter cohort of FTD patients and healthy control subjects. We compared the serum cathepsin S levels between the FTD patient groups harboring the different major genetic mutations, clinical phenotypes, or different predicted prevailing neuropathologies. Furthermore, we also compared the cathepsin S levels between FTD spectrum patients and healthy controls.

MATERIALS AND METHODS

RESULTS

Serum cathepsin S levels were first compared between the total group of FTD patients and HC. There was no statistical difference detected (p = 0.171, B = –0.058) between the groups (Fig. 1A). Sex or age did not have a statistically significant effect on cathepsin S levels in the total study cohort (p = 0.218, B = –0.050 and p = 0.088, B = –0.004, respectively). When examined separately, cathepsin S levels did not correlate with age (p = 0.075, r = –0.197) or sex (p = 0.584, r = –0.061) in healthy controls nor in the FTD patient group; age (p = 0.386, r = –0.073) or sex (p = 0.354, r = –0.078). When the FTD patients were subgrouped according to different clinical phenotypes, comparison of serum cathepsin S levels in bvFTD patients (n = 92) to those in HC did not reveal statistically significant differences (p = 0.453, B = –0.031) either. The same was true also when comparing serum cathepsin S levels in HC to the levels in PSP (n = 22) and nfvPPA patients (n = 22) (p = 0.177, B = –0.103 and p = 0.166, B = –0.090, respectively) (Fig. 1A). There were only 3 FTD-MND patients and 2 svPPA patients and, therefore, the data were insufficient for drawing any conclusive comparisons between HC and these clinical subgroups (Fig. 1A). Data on disease severity (Clinical Dementia Rating with NACC FTLD) was available for 127 of the 144 patients. However, cathepsin S levels did not correlate with the FTLD-CDR score in the FTD patient group (p = 0.237, r = 0.106).

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Fig. 1

Serum cathepsin S levels in FTD patients of different clinical phenotypes (A), carriership of different genetic mutations (B), and predicted underlying neuropathologies (C) and healthy controls. The Y-axes are presented using logarithmic scale.

Next, serum cathepsin S levels in GRN mutation-carrying FTD patients (n = 31) were compared to those in HC or GRN mutation non-carrying FTD patients. These analyses did not show a difference between the groups (p = 0.580, B = –0.030 and p = 0.909, B = –0.007, respectively) (Fig. 1B). Comparing C9 HRE-carrying FTD patients (n = 21) to HC or to C9 HRE-non-carrying FTD patients did not reveal any statistically significant differences in the serum cathepsin S levels either (p = 0.220, B = –0.081 and p = 0.290, B = 0.075) (Fig. 1B).

Finally, we compared the serum cathepsin S levels in patients subgrouped according to their predicted neuropathologies. Neither the FTD patients with predicted TDP-43 neuropathology (FTD-TDP; n = 53) nor those with predicted tau neuropathology (FTD-Tau; n = 26) showed differences in serum cathepsin S levels when compared to each other (p = 0.920, B = –0.009) or HC (p = 0.256, B = –0.055 and p = 0.275, B = –0.076, respectively) (Fig. 1C). Comparison of the serum cathepsin S levels in patients with predicted tau neuropathology to those with predicted TDP-43 neuropathology did not indicate any correlation even when adjusted for FTLD-CDR score (p = 0.872, B = 0.018).

DISCUSSION

In this study, our objective was to assess whether alterations in serum cathepsin S levels associate with different clinical phenotypes of FTD. In addition, we assessed if serum cathepsin S levels were changed in patients carrying the main FTD causative mutations or having predicted TDP-43 or tau neuropathologies. These analyses were done in order to evaluate if serum cathepsin S levels could be utilized to differentiate the different FTD patient subgroups from each other or from HC. However, our analyses did not reveal any statistical differences between the different groups, suggesting that altered serum cathepsin S levels do not specifically correlate with any clinical FTD subtypes, predicted TDP-43 or tau neuropathologies, or the major known causative genetic mutations in C9orf72, GRN, or MAPT.

Cathepsin S is preferentially expressed in microglia, which are key mediators of immune responses in the CNS. Microglial activation may at least partially be triggered by the abnormal accumulation of the tau or TDP-43 proteins, which are fundamental neuropathological changes in FTD patient brain and associate with neurodegeneration [22]. Activated microglia, in turn, can induce the expression and secretion of lysosomal cathepsins, especially cysteine cathepsins, including cathepsin S, during early stage of neuroinflammation [23]. Moreover, cathepsin S is released by microglia and macrophages after stimulation with inflammatory cytokines or pro-inflammatory lipopolysaccharide, which can lead to the degradation of extracellular matrix components, thereby supporting the migration of microglia to the inflammation site [23]. Cathepsins are also important regulators of the function of lysosomes, a cellular compartment whose dysfunction is associated with neurodegenerative diseases, and they also cleave the FTD-linked progranulin protein [24]. Proteolytic processing of progranulin to granulin peptides is a key regulator of progranulin function in mediating anti- and proinflammatory effects. Thus, from a biological point of view, changes in cathepsins might possess biomarker potential in FTD. However, the present results in this multi-centered international FTD cohort suggest that serum cathepsin S measurements do not provide a practical diagnostic tool for FTD or its differential diagnostics. However, future studies may reveal whether assessment of other cathepsin family members or lysosomal proteins in biofluids could be utilized as biomarkers in FTD diagnostics. For example, cathepsins B, D, and L have been implicated in different neurodegenerative disorders, including FTD, AD, and Parkinson’s disease, and therefore might represent new possible biomarker candidates worth further examination in FTD patients [25]. The strengths of this study are a reasonable sample size, multicenter data, and ultrasensitive analysis methodology. The main weakness of the study was that confounding factors affecting serum cathepsin S levels could not be assessed. Cathepsin S is also expressed in peripheral tissues e.g., in the spleen. Thus, assessment of brain-derived cathepsin S in blood samples is complicated by the fact that centrally and peripherally produced cathepsin S cannot be differentiated in the assay, which might have contributed to the negative result in the present study. Moreover, some previous reports have indicated that impaired renal function in patients with chronic kidney disease (CKD), systemic inflammation, and concomitant cardiovascular disease in CKD patients affect blood cathepsin S levels [26, 27]. Unfortunately, data on renal function or inflammatory status, which might have impacted the serum cathepsin S levels, were not available for the present study. Also, it remains unclear how the disease severity in FTD patients influences the serum cathepsin S levels. Such factors and comorbidities should be kept in mind and taken into consideration as possible when, e.g., stratifying patient groups in future biomarker studies. In addition, longitudinal sampling in the same patients would help to evaluate the effect of disease progression and severity on the examined biomarker levels.