An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice

Abstract

An orally bioavailable and blood-brain barrier penetrating analog of the kinase inhibitor K252a was able to prevent the typical motor deficits in the tau (P301L) transgenic mouse model (JNPL3) and markedly reduce soluble aggregated hyperphosphorylated tau. However, neurofibrillary tangle counts were not reduced in the successfully treated cohort, suggesting that the main cytotoxic effects of tau are not exerted by neurofibrillary tangles but by lower molecular mass aggregates of tau. Our findings strongly suggest that abnormal tau hyperphosphorylation plays a critical role in the development of tauopathy and suggest a previously undescribed treatment strategy for neurodegenerative diseases involving tau pathology.

Fig. 1.

Chemical structure and pharmacokinetics of SRN-003-556. (A and B) SRN-003-556 (A) is an orally bioavailable CNS-penetrating synthetic analog of K252a (B).

Fig. 2.

SRN-003-556 prevents okadaic acid-induced tau hyperphosphorylation in rat hippocampal slices in a dose-dependent manner. Four hundred fifty-micrometer slices were prepared from hippocampi of adult Wistar rats in artificial cerebrospinal fluid buffer. SRN-003-556 was added 90 min before addition of okadaic acid (1 μM final concentration). After another 90 min, the slices were extracted and analyzed by Western blotting with phosphospecific antibodies: AP422, AT8, anti-pSer262, and Tau-1, which detects tau protein when unphosphorylated at residues 189–207. For normalization to total tau, blotted proteins were dephosphorylated exhaustively on the blot with excess alkaline phosphatase and reprobed with Tau-1.

Fig. 3.

SRN-003-556 delays/prevents the onset of motor deficits in JNPL3 mice. Starting at age 229 days, hemizygous P301L-tau transgenic female JNPL3Hlmc mice were treated twice daily with alternating doses of SRN-003-556 by oral gavage for 9 weeks. Control animals received the PEG 400 vehicle alone. Mice were food deprived 4 h before application. Mice received 100 μl of SRN-003-556 (3.2 mg/ml in PEG 400; 10 mg/kg) at 1000 hours and another 200 μl (20 mg/kg) at 1600 hours. Motor performance was tested in regular intervals with a set of formal staging criteria. The survival curve shows the number of animals that reached severe phenotypic stage in the equally sized control group and the treated group. The difference between the two groups was highly significant (P = 0.0033; log-rank test).

Fig. 4.

JNPL3Hlmc transgenic mice treated with SRN-003-556 have reduced levels of soluble aggregated 64-kDa hyperphosphorylated tau species. Tau proteins were extracted from spinal cord tissue and analyzed by Western blotting with phosphospecific antibodies AP422, AT8, and anti-pS262, followed by normalization of the signals for exposure and expression/loading. (A) AP422 and AT8 antibodies detected only the 64-kDa tau species, whereas anti-pS262 also detected the normal tau species (≈55 kDa). Quantification of the blots revealed significantly lower levels of the pathological 64-kDa tau species in the treated group compared with the vehicle group by the criteria of normalized immunoreactivities with AP422 (B), AT8 (C), anti-pS262 (D), and HT7 (E). (F) Representative for all 64-kDa tau markers, AP422 signals correlated well with phenotypic stage (Spearman r, 0.8284; P < 0.0001).

Fig. 5.

Chronic treatment of JNPL3Hlmc transgenic mice with SRN-003-556 does not reduce NFT counts. (A) Comparison of representative samples (lanes 1–3) of hindbrain extracts with a standard sarcosyl-insoluble PHF preparation (lane “sarc”) by Western blotting with the human-specific phosphorylation-independent tau antibody E1 after low-speed centrifugation of crude extracts (13,000), after high-speed centrifugation (150,000), and after enrichment in sarcosyl-insoluble pellets (Sarc); arrow denotes the abnormally migrating 64-kDa tau species. (B) Development of neuropathological features spinal cords of untreated mice: neuropil threads (blue, average of scores 0–3), tangles (NFT counts, purple), and formation of phospho-epitopes of AP422 (yellow) and AT8 (green) in relation to phenotypic stage (0, unaffected; 1, mild-moderate; 2, severe). (C) Average spinal cord NFT counts in treated cohort are similar to those of vehicle controls. (D and E) NFT counts in control (D) and SRN-003-556-treated (E) mice sorted according to stage (unaffected, blue; mild-moderate, red; severe, black).