Linoleic Acid Derivative DCP-LA Prevents Tau Phosphorylation by Targeting GSK-3β

Abnormal Tau phosphorylation and aggregation into neuronal paired helical filaments and neurofibrillary tangles cause tauopathies, a class of neurodegenerative diseases, that include Alzheimer’s disease, frontotemporal dementia and parkinsonism linked to chromosome 17, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration. Glycogen synthase kinase-3β (GSK-3β) is the most critical kinase to phosphorylate Tau. We have developed the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), with cyclopropane rings instead of cis-double bonds, as an anti-dementia drug. DCP-LA serves as a selective activator of PKC and a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B). DCP-LA prevents Tau phosphorylation due to PKC -mediated direct inactivation of GSK-3β, to PKC /Akt-mediated inactivation of GSK-3β, and to receptor tyrosine kinase-mediated inactivation of GSK-3β in association with PTP1B inhibition. DCP-LA targeting GSK-3β, thus, could become a valid drug for treatment of tauopathies.


Introduction
Alzheimer's disease (AD), a neurodegenerative disease, is characterized by extensive deposition of amyloid β (Aβ) called amyloid plaque and formation of neurofibrillary tangles (NFTs). Aβ has been long thought to be the most causative factor in AD, and therefore, a variety of AD drugs targeting Aβ have been developed. No beneficial result, however, has been obtained even though Aβ was cleaned up from the brain. A current challenge, therefore, has focused upon Tau protein.
Tau, a microtubule-associated protein, is abundantly expressed in neurons of the central nervous system. Tau is upregulated during neuronal development, to promote generation of cell processes and establish cell polarity [1]. Microtubules are the tracks for motor proteins bearing intracellular cargo transport [2,3]. Tau polymerizes and stabilizes microtubules by interacting with tubulin, and modulates microtubule dynamics including axonal transport [4][5][6][7].
When hyperphosphorylated, Tau detaches from the microtubules and forms insoluble fibrils, referred to as paired helical filaments (PHFs), followed by NFTs comprising aggregation of PHFs [8,9]. Aggregation of hyperphosphorylated Tau is responsible for tauopathies, a class of neurodegenerative diseases, that include not only AD but frontotemporal dementia and parkinsonism linked to chromosome 17, progressive supranuclear palsy, Pick's disease, and corticobasal degeneration [10].

Tau phosphorylation in the AD brain
Tau from the AD brain contains eleven Ser/Thr-Pro sites, that are phosphorylated by proline-directed kinases, and nine Ser/Thr-X sites, that are phosphorylated by non-proline-directed kinases.
GSK-3β is enriched in the brain and preferentially expressed in the hippocampus.

Interaction between Aβ and GSK-3β
GSK-3β is originally in the active form. GSK-3β is inactivated by being phosphorylated at Ser9 and activated by being phosphorylated at Tyr216 [22].
Aging, inflammation, and stress also activate GSK-3β, causing an initial Tau phosphorylation, responsible for mild cognitive impairment (MCI), a preliminary group of AD.
Pyk2 binds to and activates SH2 and SH3 domain-containing proteins like Src kinases.
Lines of evidence have pointed to other pathways for regulation of GSK-3β activity.

Linoleic acid derivative DCP-LA
PKC is classified into the conventional PKC isozymes , I, II, and , the novel PKC isozymes , , , and , the atypical PKC isozymes / and , and the PKC-like isozymes and .
The primary site of action of DCP-LA is PKC . DCP-LA activates PKC selectively and directly still in the absence of DG and calcium [47]. DCP-LA binds to the PS binding/associating sites Arg50 and Ile89 in the C2-like domain of PKC at the carboxyl-terminal end and the cyclopropane rings, respectively, which are distinct from the phorbol 12-myristate 13-acetate (PMA) binding site in the C1 domain [48] (Figure 3).
DCP-LA, on the other hand, acts as a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B) through its direct interaction [49].
PKC activation or PTP1B inhibition is capable of inactivating GSK-3β each independently. In experiments using PC-12 cells, PKC overexpression and PTP1B deficiency activate Akt and inactivate GSK-3β synergistically [35]. This indicates that DCP-LA enables more efficient inactivation of GSK-3β by cooperation of PKC activation and PTP1B inhibition [35] (Figure 4).
5xFAD mouse, as an animal model of AD, is APP/presenilin 1 (PS1) double transgenic mice that coexpress five familial forms of AD mutations such as the Swedish/London/Florida 8 mutations and the M146L/L286V mutations [50]. The Aβ1-42 levels in the 5xFAD mouse brain rise in an age-dependent manner [50]. The GSK-3β activity is enhanced in parallel with Aβ1-42 rise and Tau-Ser396 phosphorylation, responsible for PHF formation, is accelerated in the hippocampus of 5xFAD mice [51]. DCP-LA suppresses the GSK-3β activation and Tau-Ser396 phosphorylation in the hippocampus of 5xFAD mice [35]. DCP-LA, thus, has the potential to restrain Tau-Ser396 hyperphosphorylation efficiently by activating PKC and inhibiting PTP1B simultaneously.

DCP-LA ameliorates cognitive disorders by facilitating hippocampal synaptic transmission
Tau hyperphosphorylation causes tauopathies. Cognitive decline in association with tauopathies including AD, however, could not be improved only by suppression of Tau phosphorylation.
Interestingly, DCP-LA protects neurons from oxidative stress-induced apoptosis by inhibiting caspase-3/-9 activation [61]. This indicates that DCP-LA could also prevent progression of neuronal loss in the AD brain.

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DCP-LA restrains Tau phosphorylation efficiently due to PKC -mediated direct inactivation of GSK-3β, to PKC /Akt-mediated inactivation of GSK-3β, and to RTK/IRS-1/PI3K/PDK1/Akt-mediated inactivation of GSK-3β in association with PTP1B inhibition. In addition, DCP-LA induces a long-lasting facilitation of hippocampal synaptic transmission, to enhance cognitive functions. Taken together, DCP-LA could not only prevent Tau phosphorylation but improve cognitive impairments associated with tauopathies.
Consequently, DCP-LA could be developed as a promising drug for prevention and therapy of tauopathies.