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Cerebrospinal fluid endo-lysosomal proteins as potential biomarkers for Huntington’s disease

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington’s disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington’s Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington’s disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.

Lysosomal-associated membrane protein-2 (LAMP2) has pivotal roles in autophagy 62 including translocation of cargo into the lumen and as a receptor in CMA [14,15].

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LAMP2 gene expression levels and total levels of LAMP2 protein have been shown 64 to be reduced and increased in . Additionally, the strongest relationship to HD brain pathology and enhanced sensitivity to clinical 147 change in early HD [48]. Disease burden score (DBS) was calculated for each HD 148 patient using the formula [CAG repeat length -35.5] × age [49]. DBS estimates 149 cumulative HD pathology exposure as a function of CAG repeat length and the time  Sample preparation 154 Measurement of peptide concentrations was performed as previously described [27], 155 which builds on the original method developed by Brinkmalm et al.[51]. However, 156 some minor modifications were introduced. In short, 50 µL CSF was mixed with 50 157 µL of an internal standard mixture containing stable isotope-labelled peptides (JPT 158 Peptide Technologies GmbH, Berlin, Germany; Thermo Fisher Scientific Inc. 159 Waltham, MA, USA), 13 C-labelled ubiquitin (Silantes, GmbH, München, Germany) 160 and bovine serum albumin (Sigma-Aldrich Co., Saint Louis, MO, USA), diluted in 50 161 mM NH 4 HCO 3 (see S1 Table). Reduction   settings are shown in S1 Table. 199 Data extraction 200 Skyline v.19.1 [53] was used to calculate and export fragment ion peak areas.

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Skyline was also used to monitor and evaluate fragment ion traces and ratios, and to 202 determine which fragment ions to include in the analysis. The ratio between tryptic 203 peptide and isotope-labelled peptide peak area was used for quantification. In total 204 48 peptides from 19 proteins, including added bovine serum albumin as a control 205 protein, were monitored. With each set of samples analysed, four quality control 206 replicates from a CSF pool were run to normalize variation between sets of samples.

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In this case the samples were split in two sets, however prepared on a single 208 occasion but analysed using PRM-MS at different points in time. The median of the 209 first set's four quality control replicates was used for normalization by dividing the 210 median of the second set's quality control median. Then the samples in the second 211 set were divided by the resulting normalization quotient (one for each peptide). As 212 multiple peptides were monitored from each protein the complexity of the data was Precision, shown in S1 Table, was monitored by analysing eight quality control 224 replicates from a CSF pool, which were run with each sample set. The precision and   Significance level was defined as p < 0.05 and tests were Bonferroni-corrected for 273 multiple comparisons when required.

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A further exploratory analysis was undertaken on the remaining 13 endo-lysosomal 275 proteins using the same hierarchical methodology outlined above.

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Participant characteristics 278 Our cohort consisted of 20 healthy controls and 60 HD mutation carriers. The HD 279 gene expansion carriers comprised of 20 premanifest and 40 manifest HD patients.

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A single premanifest participant was removed due to missing data. There were no 281 significant differences in the gender distribution (χ 2 = 0.34, p = 0.84) among the three 282 groups or CAG repeat length among manifest and premanifest HD participants. A 283 significant difference in age was observed, with both healthy controls and manifest 284 HD patients being significantly older than premanifest, because the controls were 285 recruited to span the entire age range of HD mutation carriers. As expected, there 286 were no differences between controls and premanifest individuals in TFC, TMS, cUHDRS, SDMT and SWR, but there were differences between premanifest and 288 manifest HD patients (Table 1).   when also controlling for CAG repeat length (Table 2). Furthermore, we observed no 311 significant differences when grouping together premanifest and manifest HD   Among HD gene expansion carriers, there were no significant correlations between 328 DBS and all measured analytes (Table 3) Table   331 3). Findings remained largely the same when also controlling for CAG repeat length 332 except for LAMP2 which showed a significantly association with TFC (    Composite scores were generated for each of the three components allowing for 363 their use in for subsequent analysis. Based on the protein loadings, the three 364 components were deemed to represent lysosomal hydrolases, membrane 365 binding/transfer proteins and innate immune system/peripheral proteins (PC1, PC2 366 and PC3, respectively) (Fig 3).  across disease stage, we found no significant differences in PC1, PC2 or PC3 (Fig   381   4). We observed similar findings when CAG was included in the model (S2 Table).  Table). Composite scores relating to PC1 were not significantly related to any 390 measure of clinical severity or cognition and although PC2 demonstrated a 391 significant relationship with TFC, this relationship was not present when controlling 392 for age only (Fig 5).   Table).  mutation carriers when controlling for age. Furthermore, these findings remained 411 significant when additionally controlling for CAG repeat length (Fig 6 and Table 4).
Our exploratory analysis of all the remaining endo-lysosomal proteins found no 413 significant differences in analyte concentration across disease stage (S5 Table) or 414 significant relationships with clinical measures, except for C9 and LYZ which 415 displayed significant associations with DBS (S6 Table). [56] and has been shown to be decreased in the R6/2 mouse brain [59]. It has been 482 hypothesised that an inadequate APP response to brain iron accumulation may contribute to iron homeostatic dysfunction [60]. The association between reduced 484 CSF APP and clinical worsening in this study provides some support for APP 485 dysfunction in HD and a possible impact on disease progression.