Correlations with amyloid and tau pathology in all individuals
To determine how the analysed proteins relate to CSF concentrations of t-tau, p-tau and Aβ42, each protein was correlated with the three CSF markers. Significant associations with either t-tau, p-tau or Aβ42 were found for 63 proteins (Fig. 1A, Supplementary Tables 1, 2 and 3). The strongest correlations with t-tau concentrations were shown for β-synuclein (SNCB) (Spearman rho = 0.80; p < 0.001), rabphilin-3A (RPH3A) (Spearman rho = 0.80; p < 0.001) and brain acid-soluble protein 1 (BASP1) (Spearman rho = 0.79; p < 0.001). RPH3A and SNCB also displayed the strongest correlations to p-tau concentration together with neuromodulin (GAP43) (Spearman rho = 0.78; p < 0.001). Neuronal cell adhesion molecule (NRCAM) showed the strongest correlation with Aβ42 concentration (Spearman rho = 0.33; p < 0.001), followed by neuronal pentraxin-1 (NPTX1) (Spearman rho = 0.32; p < 0.001) and voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1) (Spearman rho = 0.31; p < 0.001). Twenty-five proteins demonstrated significant correlations with t-tau and p-tau, but not Aβ42 (Fig. 1A). Among these were GAP43, cell cycle exit and neuronal differentiation protein 1 (CEND1), amphiphysin (AMPH) and phosphatidyl-ethanolamine-binding protein 1 (PEBP1), all with moderate correlations (Spearman rho > 0.6). Although the majority of correlations were positive, we also observed weak negative correlations of both t-tau and p-tau concentrations for six proteins, transmembrane protein 235 (TMEM235), GABAA receptor regulatory Lhfpl4 (LHFPL4), mitogen-activated protein kinase 8-interacting protein 2 (MAPK8IP2), protein EFR3 homolog B (EFR3B), tenascin-R (TNR) and C-C motif chemokine 22 (CCL22) (Fig. 1A).
When comparing t-tau and p-tau concentration in all individuals a strong positive correlation was observed between the two measurements (Spearman rho = 0.93; p < 0.001, Supplementary Fig. 2). The two tau concentrations did also show moderate correlations with Aβ40 concentration (Spearman rho > 0.77; p < 0.001) but not Aβ42 concentration.
Comparison of individuals grouped by CSF Aβ42/Aβ40 ratio
To explore if the associations to t-tau, p-tau and Aβ42 concentration change in the preclinical stage of AD, the individuals were dichotomised into two groups based on CSF Aβ42/Aβ40 ratio and denoted as either amyloid-positive (A+) or amyloid-negative (A-) (Table 1). The ranges of t-tau, p-tau and Aβ42 CSF concentrations in both groups are illustrated in Fig. 1B. Significant differences between the groups were found for all three markers (pAβ42=2E-42), pt−tau=1E-07, pp−tau=1E-07). Statistically significant differences in protein levels between A + individuals and A- individuals were identified for six proteins (Supplementary Fig. 3). Among these were the previously mentioned proteins GAP43, SNCB, BASP1 and RPH3A, as well as the two proteins dimethylarginine-dimethylaminohydrolase-1 (DDAH1) and aquaporin-4 (AQP4). All proteins displayed higher levels in the A + individuals compared to the A- individuals.
T-tau associations based on Aβ42/Aβ40 ratio
The majority of the 63 proteins with significant correlations with t-tau in all individuals remained significant in both the A- (61/63) and A+ (41/63) groups (Fig. 2A, Supplementary Table 1). Yet, 33 proteins demonstrated significant differences in slopes between the two groups using linear regression models (Fig. 2A, Table 2, Supplementary Table 1). Neural cell adhesion molecule L1-like protein (CHL1) displayed the largest association to CSF t-tau concentration in the A- individuals (Spearman rho = 0.80; p < 0.001), as well as a significant difference in slopes (t = 7.13; p < 0.001; R2 = 0.64) (Fig. 2B and C) while GAP43 showed the largest association (Spearman rho = 0.82; p < 0.001; t = 5.80; p < 0.001; R2 = 0.66) in the A + group. However, the most significant slope differences were seen for transmembrane protein 132D (TMEM132D) (t = 7.88; p < 0.001; R2 = 0.64) and lymphocyte antigen 6H (LY6H) (t = 7.26; p < 0.001; R2 = 0.63). Significant differences in the slopes were not found for any of the proteins with negative association to t-tau.
Table 2
Proteins displaying significant slope differences between A + and A- individuals sorted by t-tau rho.
| | | | Aβ42 concentration | T- tau concentration | P-tau concentration |
Protein name | HGNC ID | Uniprot ID | Antibody | rho | p | rho | p | p (slope) | rho | p | p (slope) |
β-synuclein | SNCB | Q16143 | HPA035876 | 0.20 | 4E-02 | 0.80 | 6E-69 | 2E-05 | 0.79 | 8E-65 | ns |
Rabphilin-3A | RPH3A | Q9Y2J0 | HPA002475 | 0.26 | 4E-04 | 0.80 | 1E-67 | 4E-07 | 0.79 | 4E-65 | 4E-03 |
Brain acid-soluble protein 1 | BASP1 | P80723 | HPA050333 | 0.21 | 2E-02 | 0.79 | 8E-66 | 2E-08 | 0.78 | 1E-61 | 5E-04 |
Neuromodulin | GAP43 | P17677 | PA5-34943 | ns | ns | 0.79 | 5E-65 | 2E-06 | 0.78 | 1E-61 | 9E-03 |
Neural cell adhesion molecule L1-like protein | CHL1 | O00533 | HPA003345 | 0.30 | 7E-06 | 0.77 | 2E-60 | 1E-09 | 0.77 | 1E-58 | 1E-05 |
Cadherin-8 | CDH8 | P55286 | HPA014908 | 0.28 | 5E-05 | 0.75 | 1E-55 | 3E-09 | 0.75 | 7E-54 | 2E-05 |
Lymphocyte antigen 6H | LY6H | O94772 | HPA077218 | 0.25 | 8E-04 | 0.75 | 2E-54 | 4E-10 | 0.75 | 4E-54 | 2E-06 |
Transmembrane protein 132D | TMEM132D | Q14C87 | HPA010739 | 0.28 | 1E-04 | 0.75 | 5E-54 | 8E-12 | 0.75 | 6E-54 | 3E-08 |
Cell cycle exit and neuronal differentiation protein 1 | CEND1 | Q8N111 | HPA042527 | ns | ns | 0.74 | 2E-53 | 4E-06 | 0.72 | 2E-48 | 8E-03 |
SLIT and NTRK like family member 1 | SLITRK1 | Q96PX8 | HPA012414 | 0.21 | 2E-02 | 0.74 | 7E-53 | 2E-08 | 0.73 | 3E-50 | 2E-04 |
Cell adhesion molecule 2 | CADM2 | Q8N3J6 | HPA010024 | 0.31 | 5E-06 | 0.74 | 6E-52 | 1E-04 | 0.73 | 1E-50 | 4E-02 |
Oligodendrocyte-myelin glycoprotein | OMG | P23515 | HPA008206 | 0.31 | 4E-06 | 0.71 | 2E-46 | 5E-06 | 0.72 | 2E-48 | 9E-04 |
Voltage-dependent calcium channel subunit alpha-2/delta-1 | CACNA2D1 | P54289 | HPA008213 | 0.31 | 3E-06 | 0.71 | 5E-46 | 9E-07 | 0.71 | 3E-47 | 5E-04 |
Neurosecretory protein VGF | VGF | O15240 | HPA055177 | 0.29 | 3E-05 | 0.71 | 2E-45 | 1E-03 | 0.71 | 3E-47 | ns |
Neuronal cell adhesion molecule | NRCAM | Q92823 | HPA061433 | 0.33 | 7E-07 | 0.70 | 1E-44 | 3E-08 | 0.72 | 2E-47 | 6E-06 |
Neurocan core protein | NCAN | O14594 | HPA058000 | 0.28 | 1E-04 | 0.69 | 5E-43 | 6E-06 | 0.70 | 1E-44 | 3E-03 |
Amyloid-like protein 1 | APLP1 | P51693 | HPA028971 | 0.31 | 6E-06 | 0.69 | 1E-42 | 6E-07 | 0.69 | 2E-43 | 1E-04 |
Extracellular matrix protein 1 | ECM1 | Q16610 | HPA027241 | 0.27 | 3E-04 | 0.69 | 4E-42 | 4E-08 | 0.68 | 2E-41 | 2E-05 |
Amphiphysin | AMPH | P49418 | HPA019828 | ns | ns | 0.69 | 6E-42 | 3E-03 | 0.68 | 2E-41 | ns |
Cholecystokinin | CCK | P06307 | HPA069515 | 0.30 | 1E-05 | 0.67 | 7E-40 | 5E-06 | 0.66 | 9E-38 | 5E-04 |
Calsyntenin-1 | CLSTN1 | O94985 | HPA012749 | 0.31 | 5E-06 | 0.67 | 3E-39 | 2E-04 | 0.67 | 2E-39 | 2E-02 |
Neurofascin | NFASC | O94856 | HPA073444 | 0.24 | 2E-03 | 0.66 | 4E-38 | 4E-06 | 0.66 | 3E-38 | 2E-04 |
Phosphoinositide-3-kinase-interacting protein 1 | PIK3IP1 | Q96FE7 | HPA002959 | 0.28 | 8E-05 | 0.66 | 4E-37 | 9E-07 | 0.65 | 1E-35 | 5E-04 |
Peptidyl-glycine alpha-amidating monooxygenase | PAM | P19021 | HPA042260 | 0.30 | 1E-05 | 0.66 | 4E-37 | 2E-04 | 0.65 | 8E-36 | 4E-02 |
Phosphatidylethanolamine-binding protein 1 | PEBP1 | P30086 | HPA063904 | ns | ns | 0.65 | 9E-37 | 3E-03 | 0.67 | 8E-39 | ns |
Dickkopf-related protein 3 | DKK3 | Q9UBP4 | HPA011164 | 0.21 | 2E-02 | 0.65 | 2E-36 | 1E-02 | 0.66 | 3E-37 | ns |
Neuronal pentraxin receptor | NPTXR | O95502 | HPA001079 | 0.27 | 3E-04 | 0.64 | 2E-35 | 3E-03 | 0.67 | 5E-39 | 4E-02 |
Vasorin | VASN | Q6EMK4 | HPA011246 | 0.21 | 2E-02 | 0.64 | 2E-34 | 5E-04 | 0.64 | 1E-34 | ns |
Neuronal pentraxin-1 | NPTX1 | Q15818 | HPA077062 | 0.32 | 1E-06 | 0.61 | 2E-30 | 2E-02 | 0.61 | 8E-31 | ns |
Multiple epidermal growth factor-like domains protein 10 | MEGF10 | Q96KG7 | HPA026876 | 0.24 | 3E-03 | 0.60 | 1E-29 | 6E-03 | 0.61 | 9E-31 | ns |
Myelin oligodendrocyte glycoprotein | MOG | Q16653 | AMAb91067 | 0.23 | 6E-03 | 0.54 | 1E-22 | 2E-04 | 0.54 | 4E-22 | 1E-02 |
Synaptotagmin-11 | SYT11 | Q9BT88 | HPA064091 | ns | ns | 0.54 | 2E-22 | 2E-02 | 0.54 | 4E-22 | ns |
Pro-opiomelanocortin | POMC | P01189 | HPA063644 | 0.22 | 2E-02 | 0.49 | 4E-18 | 2E-04 | 0.49 | 7E-18 | 7E-03 |
P-tau associations based on Aβ42/Aβ40 ratio
Similarly as for t-tau, the majority of significant associations to p-tau in all individuals were not affected by dividing the cohort based on CSF Aβ42/Aβ40 ratio (Fig. 2A and Supplementary Table 2). In the A- individuals 60/62 associations remained significant and 35/62 in the A + group. The linear regression models revealed significant differences in slopes for 24 proteins, all significant for t-tau as well (Table 2). Again, the most significant slope differences were seen for TMEM132D (t = 6.57; p < 0.001; R2 = 0.58) and LY6H (t = 5.58; p < 0.001; R2 = 0.58), which also displayed significant associations to p-tau concentration in both A + and A- individuals.
Aβ42 associations based on Aβ42/Aβ40 ratio
When dividing individuals into A + and A-, 50 proteins displayed significant associations to Aβ42 concentration in the A- individuals. However, no significant associations were found in the A + group (Fig. 2A and Supplementary Table 3). Again, NRCAM was the protein with the largest association to Aβ42 concentration, although only in the A- individuals (Spearman rho = 0.56; p < 0.001)(Fig. 2D). Linear regression models showed that there was no significant difference in slopes between A + and A- individuals for NRCAM or any other protein although a few models could explain up to 30% of the variation in protein levels (Fig. 2E, Supplementary Table 3).
Comparison of individuals grouped by CDR score
To determine if there were any differences in associations to CSF t-tau, p-tau or Aβ42 concentrations based on CDR scores, individuals were divided into two groups: CDR ≥ 0.5 (n = 57) and CDR = 0 (n = 250) (Table 1). The range of t-tau, p-tau and Aβ42 concentrations in both groups are illustrated in Fig. 1C. No significant differences between the groups were found for either CSF marker. There were also no significant differences in protein levels were observed between individuals with CDR = 0 and CDR ≥ 0.5 (data not shown).
T-tau associations based on CDR score
A total of 60 proteins displayed a significant association to t-tau in individuals with CDR = 0, all of which did also display significant associations in the A- group. Thirty-one proteins showed significant associations to t-tau concentration in the CDR ≥ 0.5 group. GAP43 showed the most significant association in individuals with CDR ≥ 0.5 (Spearman rho = 0.72, p < 0.001)(Supplementary Fig. 4A, Supplementary Table 4) but no significant difference in slopes between individuals with CDR ≥ 0.5 and CDR = 0 (Supplementary Fig. 4B). When dividing individuals into groups based on CDR score, none of the studied proteins obtained significant differences in their slopes using linear regression.
P-tau associations based on CDR score
The same 60 proteins which showed significant associations to t-tau concentration in the CDR = 0 group also had a significant association to p-tau concentration (Supplementary Table 5). In the CDR ≥ 0.5 group, 34 proteins presented significant associations to p-tau concentration. The majority (30/34) of these were the same as for t-tau associations and GAP43 was again the protein with strongest association (Spearman rho = 0.75, p < 0.001). Significant differences in the slopes were not found for any of the proteins after linear regression.
Aβ42 associations based on CDR score
Dividing individuals based on CDR score revealed 18 proteins with significant associations to Aβ42 concentration (Supplementary Table 6) in individuals with CDR = 0, all of which were also significant when dividing the individuals into A + and A-. NRCAM also showed a significant association to Aβ42 concentration in the CDR ≥ 0.5 individuals (Spearman rho = 0.47, p < 0.05). Linear regression modelling resulted in no proteins with significant difference in slopes between CDR ≥ 0.5 and CDR = 0 individuals.
Sex differences
Linear regression did reveal a significant contribution of sex for two of the 63 previously mentioned proteins, TNR and CCL22. Furthermore, levels of regulating synaptic membrane exocytosis protein 3 (RIMS3), vascular cell adhesion protein 1 (VCAM1) and chitinase-3-like protein 1 (CHI3L1), von Willebrand factor C domain-containing protein 2-like (VWC2L) and C-type lectin domain family 2 member L (CLEC2L) did also show a significant contribution of sex in the linear regression models (Supplementary Table 7). Wilcoxon rank sum tests did display significant differences on group level between females and males for RIMS3 and VCAM-1 only (Supplementary Table 7 and Supplementary Fig. 5). Further examination showed that, although different between groups, sex had no significant interaction with the CSF markers for any of the seven proteins (Supplementary Table 7).
NfL, NRGN and APOE ε4 carrier status
The obtained protein profiles were furthermore investigated in relation to NfL and NRGN, two of the suggested markers of neurodegeneration and synaptic dysfunction, as well as APOE ε4 carrier status. The measured NfL concentrations did not display strong correlations to any of the other suggested markers for AD, neurodegeneration or synaptic dysfunction (Supplementary Fig. 2). Weak significant correlations to NfL concentration was found for neurofilament medium chain (NEFM), glutamine synthetase (GLUL) and glutamate decarboxylase 1 (GAD1) (Pearson R = 0.23–0.30; p < 0.01) (data not shown). No significant differences could be observed when comparing NfL concentration between A + and A- individuals. However, upon stratifying individuals into groups based on both CSF Aβ42/Aβ40 ratio and NfL concentration, nine proteins displayed significant differences between groups (Fig. 3 and Supplementary Fig. 6). Two trends could be identified among the nine proteins, i) higher protein levels in Nf + individuals, independently of Aβ42/Aβ40 ratio and ii) higher protein levels in the Nf + A + group. The NEFM protein was found at higher levels in Nf + individuals (Fig. 3A) whereas GAP43 mainly displayed higher levels in the Nf + A + individuals (Fig. 3B). Nonetheless, the dichotomization did not contribute to any new trends in the interaction between group and the CSF markers (data not shown).
The measured NRGN concentration showed moderate to strong correlations with Aβ40, t-tau and p-tau concentration (Supplementary Fig. 2). Correlating the 104 analysed protein profiles to NRGN concentration revealed 71 proteins with significant associations, of which 37 displayed a Pearson R > 0.5 (Supplementary Fig. 7A). A significantly higher NRGN concentration was identified in the A + individuals compared to the A- individuals (p < 0.001, Supplementary Fig. 7B).
NEFM was the only protein that displayed a significant difference in protein levels between APOE ε4 carriers and non-carriers (p < 0.01, data not shown), a difference that was observed again when combining APOE ε4 status and CSF Aβ42/Aβ40 ratio to divide individuals into groups. NEFM levels were higher in APOEε4 + individuals independently of CSF Aβ42/Aβ40 ratio (Fig. 3C). GAP43 instead showed a trend of higher levels in A + individuals regardless of APOE ε4 carrier status, although the difference was not statistically significant (Fig. 3D). Dichotomization of individuals based on CSF Aβ42/Aβ40 ratio and APOE ε4 status did not affect the interaction between group and the CSF markers (data not shown).
Tissue expression and regional variation in the brain
To characterize the proteins included in this study we looked further into the variation in RNA expression of the corresponding genes in human tissues, using tissue expression profiles available in the Human Protein Atlas (HPA) (33). When comparing the expression levels across 37 different human tissues, the genes separated into four clusters. One cluster with more general expression in all tissue types (Cluster 1), one with elevated expression in the liver compared to other tissues (Cluster 2), one with higher expression in the brain (Cluster 3) and one last mixed group with high expression in brain or other tissue types (Cluster 4) (Supplementary Fig. 8). The majority of the studied proteins were found in the cluster with high expression in the brain and lower expression in the remaining tissues (Cluster 3). Cluster 2 contained none of the proteins for which significant correlations were observed.
Clustering of gene expression based on regional brain profiles (HPA Brain Atlas) (34), showed brain region clustering of the forebrain versus brainstem (Fig. 4A). Gene expression clusters identified visually included genes with a lower expression in the cerebellum compared to other brain regions and slightly higher expression in brainstem regions compared to forebrain (Cluster 1). Many of these genes were glia-related and especially associated to oligodendrocytes, which explains the correlation to white matter rich regions of the brain stem. Additionally, a few genes with lower expression in the white matter rich regions corpus callosum and thalamus could also be identified in a cluster (Cluster 2) and included neuronal markers such as NEFM, microtubule-associated protein 2 (MAP2), BASP1 and GAP43. The only examples of genes that were classified as regionally elevated, defined as a 4-fold higher expression level in one region compared to all other regions, were pro-opiomelanocortin (POMC) (hypothalamus), potassium voltage-gated channel subfamily C member 1 (KCNC1, cerebellum), proenkephalin-B (PDYN, basal ganglia) and cholecystokinin (CCK, forebrain regions). In summary, very few genes could be classified as regionally elevated within the brain and no clear trends or clustering based on correlations to the core AD biomarkers were found.
No clear differences regarding protein location or cellular specificity were observed when comparing the available spatial protein information by looking at the IHC-images available at the Human Protein Atlas (www.proteinatlas.org). Examples of neuronal as well as glial proteins were identified independent of the observed correlations to Aβ42, t-tau or p-tau, as well as proteins detected in the neuropil. Even so, three representative proteins (RPH3A, AMPH and TNR) were selected for immunohistochemical staining in order to confirm their spatial distributions and compare patterns across proteins with different association profiles in normal brain tissue as well as AD patients (Fig. 4B). All three selected proteins displayed a similar staining pattern in the cerebral cortex and hippocampal formation, detected in subsets of neuronal cell bodies and with general neuropil positivity. However, the detailed location in cerebellum differed between the three proteins. RPH3A demonstrated positivity of interneurons in the molecular layer as well as neuropil positivity. AMPH showed positivity in neuropil and synaptic connections in the granular layer, while TNR mainly showed neuropil positivity. The proteins displayed a similar general neuropil positivity in the AD tissue, but TNR also showed positivity associated to the plaques which AMPH and RPH3A did not.