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Alzheimer’s disease skin fibroblasts selectively express a bradykinin signaling pathway mediating tau protein Ser phosphorylation ¹

Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri, USA

²Correspondence: Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8108, St. Louis, MO, 63110, USA. E-mail: baenzign{at}thalamus.wustl.edu

SPECIFIC AIM

This study is based on two underlying concepts: 1) genetic forms of Alzheimer's disease (AD) are autosomal dominant, a profile generally associated with toxic gain of function; 2) bradykinin (BK), a neuropeptide mediator found in brain and peripheral tissues, is generated under conditions known as risk factors for AD, including stroke and traumatic head injury. BK B₂ subtype receptors show the same spatial distribution in brain as does the pattern of earliest lesions found in AD. The aim of this study was to determine whether an accessible peripheral tissue—skin fibroblasts from AD patients or at-risk persons—demonstrates a gain of function associated with the BK receptor pathway and relevant to AD pathogenesis.

PRINCIPAL FINDINGS

1. PKC activation by phorbol ester increases I/L B₂ BK receptors in AD skin fibroblasts
In established human fetal lung fibroblast models of cellular aging, BK B₂ receptors adopt alternative biologically active molecular forms due to differential receptor phosphorylation. These modified receptors, termed intermediate (I, K_d 5 nM) and low (L, K_d 42 nM) affinity, display gain of function for PGE₂ production and are evoked by phorbol ester activation of protein kinase C (PKC). We now show that skin fibroblasts of patients with AD developing around age 35–50 due to mutations in presenilins (PS-1, PS-2) show up to a 451% increase in I/L modified BK B₂ receptors when treated for 1–2 min with 25 nM phorbol myristate acetate (PMA), as determined by immunoblotting with mAbs selective for the I/L modified receptor forms. This increase is blocked by the PKC inhibitor GF109203X (bis-indoleylmaleimide) at 240 nM. In contrast, I/L BK B₂ receptors in normal age-matched control fibroblasts occur only at trace levels that do not increase with PKC activation.

2. BK B₂ I/L receptors are increased by PKC activation in Trisomy 21 fibroblasts
PMA activation of PKC also rapidly increases BK B₂ I/L receptors in skin fibroblast cell lines from two unrelated persons with Down syndrome (Trisomy 21) aged 19 and 20, who are likely to develop AD by age 35–45 due to excess gene dosage of the APP precursor. PMA treatment increases I/L modified BK B₂ receptors to 231 ± 35% and 292 ± 47% of the PMA-untreated baseline I/L receptor levels in the respective cell lines, whereas a similarly treated age-matched cell line from a normal 17-year-old shows no increase in I/L forms of B₂ BK receptors at all.

3. BK itself increases the amount of I/L BK B₂ receptors in AD skin fibroblasts via PKC
BK itself activates PKC by B₂ receptor-mediated signaling. In PS-1 mutant AD and Trisomy 21 skin fibroblasts, 250 nM BK causes a rapid 2.5-fold elevation of I/L BK B₂ receptors not seen in normal skin fibroblasts from individuals of any age. This increase in I/L BK B₂ receptors due to BK is blocked by the PKC inhibitor GF109203X (Fig. 1 , w/GF) and by the B₂ receptor antagonist Thi^5,8-D-Phe⁷-BK (Fig. 1 , w/a-B2), indicating that the generation of the I/L forms is occurring via BK receptor-mediated activation of PKC. An additional AD skin fibroblast cell line derived from a 77-year-old AD patient having no mutations in either PS-1 or PS-2 (denoted non-presenilin in Fig. 1 , n-PS) shows similarly increased I/L BK B₂ receptors upon BK-mediated activation of PKC. No increase in I/L receptors occurs in normal control fibroblasts upon PKC activation by BK.

View larger version (33K): [in this window] [in a new window]   Figure 1. BK B2 receptor activation increases BK B2 I/ L receptors in AD and AD-susceptible fibroblasts via a PKC-mediated response. Skin fibroblasts from normal, AD, and Trisomy 21 individuals were treated with 250 nM BK for 1 min and analyzed by quantitative immunoblotting with mAbs against I/L BK B2 receptors and densitometry of the receptor bands. Values represent mean ± SE for Trisomy 21 (shaded bar, n=4); PS-1A246E (black bars, left to right representing cells treated with BK alone (n=11) and with BK in the presence of PKC inhibitor GF109203X (w/GF) or a B2 receptor antagonist (w/a-B2), n=3 each); non-presenilin AD skin fibroblasts (horizontal striped bar, n=5); and age-matched controls (clear bar, n=7).

4. BK causes selective tau protein phosphorylation in PS-1 AD and Trisomy 21 fibroblasts via a PKC-mediated mechanism
AD skin fibroblasts display signal transduction through the increased I/L BK B₂ receptors that targets the tau protein specifically involved in AD pathology. AD and normal skin fibroblasts bear equivalent amounts of tau protein, visualized at M_r 55 and 62 kDa by immunoblotting with anti-tau antibody (Fig. 2 , see lane marked "T"). BK increases Ser phosphorylation of tau protein (Fig. 2 , see lanes marked "0–10") in both AD and Trisomy 21 skin fibroblasts. The major BK stimulatory effect at 250 nM BK represents the concentration range characteristic of I/L modified BK B₂ receptor biologic activity. BK-mediated tau Ser phosphorylation increases by 256% in BK-stimulated AD fibroblasts compared with unstimulated AD cells and by 251% in BK-stimulated Trisomy 21 fibroblasts compared with non-BK-treated cells. Increased Ser phosphorylation of tau in the BK-responsive AD and Trisomy 21 skin fibroblasts is blocked by pretreatment with the PKC inhibitor GF109203X. BK does not induce Ser phosphorylation of tau protein in normal age-matched control fibroblasts.

View larger version (23K): [in this window] [in a new window]   Figure 2. BK rapidly stimulates tau Ser phosphorylation in presenilin mutant AD and AD-susceptible Trisomy 21 fibroblasts in a PKC-dependent manner. Fibroblasts were treated with 250 nM BK for the time points shown in a representative experiment each for Trisomy 21 and PS-1(M146L) cells. Non-ionic detergent lysates of cells were immunoblotted with anti-bovine tau antibody (T lane) and with anti-phospho Ser antibody [lanes corresponding to no BK (0) through 5 and 10 min of BK treatment].

CONCLUSIONS AND SIGNIFICANCE

Our studies demonstrate that persons who have or are likely to develop AD have altered BK receptor signaling detectable in a peripheral tissue, their skin fibroblasts, compared with normal persons. This alteration in signaling recruits a phosphorylation cascade that targets a major protein recognized in the molecular pathogenesis of AD, the tau microtubule-associated protein as well as BK B₂ subtype receptors (Fig. 3 ). A transient burst of PKC activity stimulated by BK itself or other routes of activation mobilizes this cascade to increase the cellular content of alternatively phosphorylated BK B₂ receptor I/L affinity forms that are biologically active in the BK concentration range of 25–250 nM. The result is tau protein phosphorylation on Ser residues, which comprise the known early loci for tau hyperphosphorylation during AD disease progression. Initial PKC activation by BK B₂ receptors themselves consistently increases I/L BK B₂ receptors in skin fibroblasts representing AD arising from presenilin mutations, Trisomy 21, and a familial but non-presenilin-mediated AD disease process. This BK-operated signaling cascade is not found in skin fibroblasts from normal individuals from ages 17–82.

View larger version (26K): [in this window] [in a new window]   Figure 3. Alzheimer's disease fibroblast BK responses report an altered intracellular milieu. In this schematic summary of altered signaling in AD human skin fibroblasts, the BK B2 receptor model shown at left bears cytoplasmic domain phosphorylation sites that can be modified to generate the I and L biologically active affinity forms. This phosphorylation (P) occurs when the kinase cascade K1 is activated by the BK receptor or by other routes to PKC activation (center receptor model). The modified receptors recruit the kinase cascade K2, which results in Ser phosphorylation of tau (receptor model at right). Kinase cascades K1 and K2 include at least PKC and likely additional kinases that may overlap between cascades 1 and 2. Scaffold proteins (Sc) may serve to link I/L modified BK B2 receptors to the kinase cascades. The nature of these additional participants remains to be defined: to identify the component or components that represent the root cause of enhanced signaling via this cascade in AD skin fibroblasts. In control human skin fibroblasts from donors of any age the initial modification of BK B2 receptors to yield I/L modified forms takes place only at a trace level, which is not PKC-dependent and does not progress to tau Ser phosphorylation.

Enhanced I and L affinity BK B₂ receptors and consequent tau Ser phosphorylation can be detected in Trisomy 21 skin fibroblasts decades before involvement of the brain reaches a level triggering the onset of symptomatic AD at age 35–45. The PKC-mediated increase in skin fibroblast I/L BK B₂ receptors is detected in symptomatic AD reflecting mutations at the 2nd (M146L) and 6th (A246E) transmembrane domains and large cytoplasmic loop (L286V) of PS-1, as well as the homologous 2nd transmembrane domain mutation N141I of PS-2. Further studies are slated to determine the expression of this signaling cascade in skin fibroblasts from younger relatives of the PS-1 AD probands well before the predicted age of disease onset.

The role of amyloid peptide Aß_1-42, another major molecular entity associated with AD, has been advanced as the causative factor in the disease process. Genetically based AD and Trisomy 21 skin fibroblast cell lines as well as those from sporadic AD patients display variably elevated levels of intracellular and secreted Aß_1-42. Parameters related to Aß_1-42 have been proposed as an AD screening tool using accessible peripheral tissues. However, recent serious complications in clinical approaches toward AD management based on immunization to help clear this peptide indicate that Aß_1-42-centered strategies alone may not suffice in designing risk assessment, prevention, and treatment modalities for AD. Furthermore, transgenic animal models of AD generated by manipulation of Aß metabolism fail to reproduce the tau pathology seen in the human disease. The altered BK receptor signaling cascade evident in primary human skin fibroblasts (Fig. 3) represents a molecular signature that is able to identify an AD patient in these studies known not to have any PS-1 or PS-2 mutations and whose Aß_1-42 status is undetermined. The phosphorylation cascade embodied in this molecular signature includes kinases and phosphatases that not only interact with tau protein but may drive the early synaptic loss and neuronal death associated with abortive cell cycle re-entry in AD. The adverse outcome of Aß immunization clinical trials with humans and experimental animal models for AD suggests that dysregulation of phosphorylation cascades such as the BK-mediated pathway we define here deserves renewed attention in discussions about the molecular pathogenesis as well as the detection of this disorder.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1147fje; doi: 10.1096/fj.02-1147fje

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This Article Full Text (PDF) All Versions of this Article: 17/15/2319 02-1147fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JONG, Y.-J. I. Articles by BAENZIGER, N. L. Search for Related Content PubMed PubMed Citation Articles by JONG, Y.-J. I. Articles by BAENZIGER, N. L.