Keratin 9 Expression and Correlations with Other AD-associated Proteins

Keratin 9 expression levels were examined in blood plasma samples from a cohort comprising 60 healthy individuals and 58 AD patients, as shown in Fig. 1A. The actual concentrations observed in this cohort were lower than those obtained for an alternative cohort in a previous study16, however, the trends observed were analogous, with expression levels decreasing in healthy individuals (Mean = 34.7 ± 47.6 pg/ml) when compared to AD patients (Mean = 64.4 ± 88.0 pg/ml). The variation in concentrations observed between the different studies may be due to the use of different anticoagulants during blood collection, a factor which is known to affect metabolite concentrations34. Whilst still not reaching significance, the p-value of the change due to AD decreased for the larger cohort (p = 0.1399; n = 58 AD/60 Healthy) indicating that it may continue to improve as the sample size increases. In Fig. 1B, there appears to be some level of association between blood plasma Keratin 9 concentration and age in healthy individuals, (r = −0.2316; p = 0.075) which is dysregulated in the AD cohort (r = 0.1646; p = 0.2168).

Figure 1 Keratin 9 expression in blood plasma in healthy and AD patient cohorts (A) and the correlation of these concentrations with the age of healthy (B(i)) and AD (B(ii)) individuals. Full size image

In order to gain an understanding of the role of Keratin 9 in AD, interactions with other AD-associated proteins were sought. Immunoassays of Aβ42, ApoE, Clusterin, Fibrinogen, SPARCL1 and Tau15,16,35 were undertaken on blood plasma samples from the two patient cohorts. The concentrations of these proteins are detailed in Table 2. It should be noted that, whilst not the focus of this study, a significant difference (p = 0.0063) between the concentration of fibrinogen in the healthy (49.59 ± 20.72 μg/ml) and AD (41.57 ± 21.72 μg/ml) patient samples was observed lending weight to previous studies which identify it as a potential biomarker of AD36. Additionally, the correlations between age and concentration of ApoE, SPARCL1 and Tau were found to be disrupted upon onset of AD whilst the correlation between Aβ42 and age strengthened in the AD cohort when compared to healthy individuals (Table 2).

Table 2 Blood plasma concentrations of putative AD biomarker targets as measured by immunoassay. Full size table

When the relationship between concentrations of Keratin 9 and Aβ42, ApoE, Clusterin, Fibrinogen, SPARCL1 and Tau were examined (Table 3), significant correlations were demonstrated between blood plasma concentrations of Keratin 9 and ApoE, Clusterin or Tau. The strength of all these correlations was found to increase in the AD cohort when compared to the healthy individuals. A correlation with SPARCL1 was observed, but the strength of this association remained unchanged between the two cohorts. No correlations were identified between blood plasma concentrations of Keratin 9 and Aβ42 or Fibrinogen in either the healthy or AD cohorts (Table 3).

Table 3 Correlations between protein concentrations of Keratin 9 and other AD-associated molecules in blood plasma samples. Full size table

Before speculating on the meaning of these results, the possibility of contamination was also addressed. Due to the expression of Keratin 9 in the outer, terminally differentiated epidermis, it is present in shed skin, which is a major component of dust. However unlikely, it is conceivable, that results from some previous studies could have attributed dust contamination to the detection of Keratin 937. Furthermore, taking blood and CSF samples involves puncturing the skin, and as such may be a route through which keratinocyte components could enter the clinical samples. ELISA measurements of Keratin 9 in both AD patients and healthy controls collected within this study suggest that there is some level of expression in both cohorts (Fig. 1A). Whilst the possibility of contamination causing these baselines levels of expression cannot be disregarded, it cannot account for studies where differences in expression levels have been identified in identically handled samples. Work in our laboratory has previously identified the presence of Keratin 9 in AD CSF but not in normal CSF16 substantiating the significance of our findings and suggesting that there is a biological basis behind the results as opposed to contamination.

Initial in silico Analysis

The data outlined above suggests that Keratin 9 may be implicated in the mechanistic pathways underlying AD. To further ascertain the nature of association, in silico protein association studies were undertaken using VisANT software, as described in Methods. Initially, the known interacting partners of Keratin 9 were examined with the interactome produced by these molecules illustrated in Fig. 2. This identified 54 proteins that have been experimentally proven to interact with Keratin 9, which are listed in Table 4, along with the experimental methods used to ascertain these interactions. Current knowledge of the function of Keratin 9 is limited; it has been shown to provide scaffolding to cells3 and act as an epidermal differentiation marker38. This VisANT analysis implies it may have further complex roles as it interacts with proteins that possess a wide range of functions (Table 4). The names and details of these proteins are listed in Table 4. Additionally, the interactome produced substantiates recent findings that Keratin 9 may have alternate expression sites to its well-characterised expression in palms and soles. For example, Keratin 9 has been shown to interact with IQCB139, which is localized in cilia of renal epithelial cells and, interestingly photoreceptor cells40. This adds weight to the recent discovery that Keratin 9 can be found in the proteome of tears8. One of the most important implications of this interactome is that it provides further validation of the presence of Keratin 9 in blood plasma10,16 as it can interact with five of the most abundant proteins in blood serum: Albumin, Apolipoprotein A-I and the heavy chain constant regions of Immunoglobins A, G and M41.

Figure 2: A basic Keratin 9 interactome determined using VisANT software. The interactome comprises the 54 proteins demonstrated to interact directly with Keratin 9 with the lines linking each node colour coded according to method used to establish the interaction. Further information on the nodes and interactions can be found in Table 4. Full size image

Table 4 Components of the Keratin 9 interactome determined using VisANT software. Full size table

Further in silico Analysis: Expanding the Keratin 9 Interactome

Although this interactome provides a useful start point from which to understand the involvement of Keratin 9 in AD, it only details direct molecular interactions; expanding the interactome would highlight further biochemical pathways that may influence/be influenced by variations in Keratin 9 expression. With this in mind, we examined the additional interactions of all 54 identified Keratin 9 interacting proteins. However, simply expanding every node resulted in high levels of complexity in the outputted data. We proceeded, therefore, to examine the interactions between Keratin 9 and other molecules known to be involved in AD pathology directly rather than indirectly. VisANT was utilised to produce an interactome detailing the interactions of Keratin 9 with APP, Tau (MAPT) and ApoE, three molecules with a longstanding association with AD (together, referred to as the AMA interactome). Molecules found to have two or more interacting partners within the derived network (i.e. they interact with at least two out of ApoE, APP, Keratin 9 and Tau) are included in the resultant interactome (Fig. 3). Correlations between expression levels of Keratin 9 and those of ApoE and Tau were earlier found to be disrupted in blood plasma samples from AD patients (Table 3). Whilst Keratin 9 has not yet been demonstrated to interact directly with APP, ApoE or Tau, Fig. 3 demonstrates that it is closely connected to all three molecules, requiring only one bridging molecule. The molecules found to interact with Keratin 9 and the AMA interactome are summarised in Table 4. In the case of ApoE Keratin 9 is linked via either VCAM1, ALB or UBC whilst for Tau it is linked via UBC and YWHAQ and for APP, it is linked via ALB, APOA1, COPS5, CUL3, CUL4B, GABARAPL2, GRB2, IGHM, MAP1LC3A, MDM2, NEDD8, PIK3R2, SHC1, TANK, TRAF3IP1, UBC and UCHL5. These molecular associations fail, however, to shed much more light on the problem since it is unlikely that Keratin 9 levels would affect APOE, MAPT or APP and vice versa through such a short pathway. Furthermore, many of the linking molecules, such as UBC, are ubiquitously expressed and interact with a broad range of proteins so any effect manifesting through these molecules would not explain the specificity of AD symptoms. It is therefore necessary to expand the interactome network: a series of several, more specific interactions that link these proteins is potentially the key to solving the problem as opposed to searching for the shortest connection paths. One way to solve this problem, whilst avoiding generating too much complexity in the interactome, is to place Keratin 9 in the context of pathways that are already thought to play a role in AD.

Figure 3: The Interactome of Keratin 9, Apolipoprotein E (APOE), Tau (MAPT) and Amyloid Precursor Protein (APP) (nodes shown in red). VisANT software was used to identify all interacting molecules. Interacting molecules are identified as green (interacting with 2 proteins), yellow (interacting with 3 proteins), or blue (interacting with 4 proteins). Proteins with only a single interaction were excluded from the Interactome. Only the nodes with direct links to Keratin 9 have been labelled. Full size image

In previous work we have used Reactome (http://www.reactome.org/)25,42 to identify pathways in which AD biomarkers may be participating25. Initially, the same approach was implemented here whereby Keratin 9 and all 54 of its interacting molecules that were identified through VisANT were inputted into Reactome. However 10 out of these 55 molecules, including Keratin 9, were not found in the Reactome database, and as a consequence this method was deemed inappropriate for determining the pathways associated with Keratin 9. Instead, the functions of each of the Keratin 9 interacting proteins were evaluated using the UniProtKB database, which displays the complete Gene Ontology annotation as well as a full list of Reactome entries for each protein. Additionally, since VisANT only displays proteins that share physical interactions, we searched the literature for evidence of transcriptional regulation of Keratin 9 to establish whether Keratin 9 regulators play a role in AD. As a result of this, several pathways linked to these interacting proteins and regulatory proteins were highlighted, including wnt signalling and the ubiquitin proteasome system which have already been linked to AD43,44,45.

Wnt Signalling in AD

The canonical wnt signalling pathway regulates the expression of a wide range of genes throughout the body and has been strongly implicated in AD43,44. The cascade is initiated when wnt ligands complex with LRP5/6 and Frizzled (Fz) receptors. Fz then activates Dishevelled protein, which forms a complex with GSK3β, adenomatous polyposis coli (APC), Axin and beta-catenin resulting in inactivation of GSK3β and stabilization of beta-catenin. Beta catenin can then bind to T cell receptor (Tcf)/Lymphoid enhancer-binding factor (LEF) transcription factors by displacement of the repressor protein, Groucho leading to activation or repression of target genes. In the absence of wnt, or in the presence of a wnt antagonist, GSK3β remains active, facilitating phosphorylation of beta-catenin, which is targeted to the ubiquitin-proteasome degradation pathway44. This prevents beta-catenin from entering the nucleus and interacting with transcription factors. It is thought that downregulation of components of this cascade can result in many of the key changes that occur in the AD brain: repression of wnt signalling has so far been linked to the processing of APP, Aβ peptide neurotoxicity and tau phosphorylation43. For example beta secretase (BACE1), the APP cleaving enzyme, is suppressed following wnt activation via beta-catenin binding to TCF4, a BACE1 promoter46. Therefore, downregulation of the wnt pathway can result in increased cleavage of APP and therefore increased Aβ levels.

VisANT analysis suggests that Keratin 9 may be linked to this cascade via interactions with APC and Cadherin 1 (CDH1), which interacts with β-catenin in adherens junctions. It is unlikely however, that Keratin 9 could affect this pathway through binding to CDH1 or APC as there is no evidence of Keratin 9 acting as a signalling molecule. It is more likely that Keratin 9 levels could be influenced by this pathway as it has been suggested that wnt signalling may be involved in keratinocyte development. Dickkopf 1 (DKK1), an antagonist of the wnt pathway, has been shown to induce the expression of Keratin 9, resulting in a thicker epidermis47. Furthermore, increased expression of DKK1 has been seen in brain tissue of AD patients and this has been causally linked to the neurodegeneration associated with the disease48. It is thought that expression of Aβ induces expression of DKK1 in a P53 dependent manner and conversely, repressing DKK1 reduces Aβ toxicity49. Increased expression of DKK1 could therefore not only help explain the characteristic amyloid beta toxicity, but also the presence of Keratin 9 in the CSF of AD patients. Although, to our knowledge, this is the first time a link has been proposed between DKK1 and Keratin 9 in non-epidermal tissue, both DKK1 and Keratin 9 have separately been shown to have diagnostic potential for hepatocellular carcinoma due to their dysregulation in blood serum10,50. This suggests that, in certain disease conditions, DKK1 could induce the expression of Keratin 9 in non-epidermal tissue.

Interestingly, our results also show a correlation between changes in Keratin 9 and Clusterin levels in AD (Table 3). This could be explained by previous research by Killick and colleagues49, which suggests linkages between Clusterin, DKK1 and Aβ. They found that knockdown of Clusterin expression results in reduced Aβ toxicity and DKK1 expression, suggesting that Aβ induced DKK1 expression is dependent on Clusterin, as well as P53. They also highlighted a potential mechanism, thought to be the amyloid cascade, whereby Clusterin mediates neurotoxicity in an Aβ induced clusterin/P53/DKK1/wnt-PCP-JNK pathway. Since our immunoassay detection did not include DKK1 or P53, we can only speculate that these may be linked to Clusterin and Keratin 9 based on previous research. It would therefore be of interest to include DKK1 and P53 in our biomarker panel in future in order to establish for ourselves whether these correlate with Clusterin, Aβ or Keratin 9.

Keratin 9 and Blood Brain Barrier Dysregulation in AD

The presence of Keratin 9 in CSF has previously been demonstrated using various mass spectrometry techniques12,15 and 2D electrophoresis13, but these techniques gave no indication of the concentration at which it existed. To our knowledge, the first immunoassay validation of the presence of Keratin 9 in CSF was obtained during our previous study which demonstrated the presence of Keratin 9 in the CSF of AD patients but not in healthy individuals16. Keratin 9 has also been identified in the proteome of blood plasma multiple times41,51,52,53,54,55,56,57,58,59,60,61,62. Although sample contamination cannot be ruled out, and we can only speculate on why Keratin 9 is present in blood, the sheer number of studies documenting this result adds plausibility to our finding of Keratin 9 in blood plasma in both healthy and AD patients. Furthermore, our finding that Keratin 9 was present in the CSF of AD individuals but not in healthy individuals16, could suggest that there are differences in the blood brain barrier (BBB), enabling Keratin 9 to pass from the blood into CSF in AD individuals.

Dysfunction of the BBB has already been strongly implicated in AD. The integrity of the BBB is compromised in AD due to down-regulation of Claudin proteins, resulting in destabilisation of tight junctions between endothelial cells63. Evidence also suggests that p-glycoprotein, an Aβ transporter at the BBB, is down-regulated in AD, resulting in compromised Aβ clearance and accumulation of Aβ in the brain64. Interestingly, expression of p-glycoprotein and maintenance of tight junctions in the BBB are known to be regulated by the wnt signalling pathway63,65. Increased expression of DKK1, as mentioned previously, inhibits this pathway and therefore contributes to increased BBB permeability, decreased p-glycoprotein expression and Aβ accumulation. Increased permeability of the BBB could therefore enable Keratin 9, which is up-regulated by DKK147, to pass from blood to CSF in AD patients.

Keratin 9 and the Ubiquitin Proteasome System in AD

The interactome produced using VisANT and subsequent analysis revealed that at least 12 of the Keratin 9 interacting partners: CAND1, CBL, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, DCUN1D, MDM2, NEDD8 and UBC, are components of the Ubiquitin Proteasome System (UPS)66,67,68,69. This suggests that Keratin 9 may be ubiquitinated and targeted for degradation by the proteasome. However, the abnormal presence of Keratin 9 in CSF and the elevated levels of Keratin 9 in blood of AD patients indicate a failure in this system, which prevents Keratin 9 degradation. Dysregulation of the UPS has previously been linked to AD as a significant decrease in proteasome activity has been observed in various regions of the AD brain and this is thought to result in the aggregation of Aβ into plaques45. This may also explain the build-up of Keratin 9 and therefore Keratin 9 may be a useful biomarker to detect the UPS dysregulation associated with AD.

Keratin 9: More than just a structural molecule

In addition to functioning as scaffolding proteins within cellular cytoskeletons, some keratins are known to be involved in cell signalling, communicating with extracellular matrix (ECM) components and other cells through desmosomes and hemidesmosomes70,71. Although Keratin 9 has not yet been implicated in any signalling pathways, VisANT analysis reveals interactions with several cytoskeletal and ECM components, implying a potential signalling role. Specifically, Keratin 9 interacts with CDH1 and ITGA4, which are involved in cell-cell junctions and cell-matrix interactions, and FN1, a component of the ECM. Keratin 9 also interacts directly with other cytoskeletal components, including KRT1 and indirectly with microtubules through interactions with MAP1LC3A and MAP1LC3B. There is a wide range of other proteins with which Keratin 9 also interacts (Table 4) for which the implications of the interactions are currently unclear. As these proteins have a variety of functions it could be speculated that the interactions may have unforeseen consequences for other downstream pathways. If this were to be proven and if the affected pathways influence AD it would mean that dysregulation of Keratin 9 expression, which currently appears to be a consequence of AD, may have consequences of its own that may contribute to AD onset and progression.