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.
Article: Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.[show abstract] [hide abstract]
ABSTRACT: The microtubule-associated protein tau is a major component of the paired helical filaments (PHFs) observed in Alzheimer's disease brains. The pathological tau is distinguished from normal tau by its state of phosphorylation, higher apparent M(r) and reaction with certain antibodies. However, the protein kinase(s) have not been characterized so far. Here we describe a protein kinase from brain which specifically induces the Alzheimer-like state in tau protein. The 42 kDa protein belongs to the family of mitogen activated protein kinases (MAPKs) and is activated by tyrosine phosphorylation. It is capable of phosphorylating Ser-Pro and Thr-Pro motifs in tau protein (approximately 14-16 P1 per tau molecule). By contrast, other proline directed Ser/Thr kinases such as p34(cdc2) combined with cyclin A or B have only minor effects on tau phosphorylation. We propose that MAP kinase is abnormally active in Alzheimer brain tissue, or that the corresponding phosphatases are abnormally passive, due to a breakdown of the normal regulatory mechanisms.The EMBO Journal 07/1992; 11(6):2131-8. · 9.20 Impact Factor
Article: Assembly of tau in transgenic animals expressing P301L tau: alteration of phosphorylation and solubility.[show abstract] [hide abstract]
ABSTRACT: Transgenic mice (JNPL3), which develop neurofibrillary degeneration and express four-repeat human tau with P301L missense mutation, were characterized biochemically to determine whether the development of aggregated tau from soluble tau involves an intermediate stage. Homogenates from mice of different ages were separated into buffer-soluble (S1), sarkosyl- and salt-extractable (S2) and sarkosyl-insoluble pellet (P3) fractions, and analyzed for human tau distribution, phosphorylation and filament formation. S1 and S2 fractions contained 50-60-kDa tau whereas the S2 fraction also had 64-kDa tau. The level of tau in the P3 fraction increased in an age-dependent manner and correlated positively with the soluble tau concentration. The P3 fraction from 2.5-6.5-month-old mice contained 64- and 50-60-kDa tau, whereas that from 8.5-month and older transgenic animals contained mostly 64-kDa and higher molecular weight tau. The S2 and P3 fractions contained comparable amounts of 64-kDa tau. The 64-kDa tau was predominantly human, and phosphorylated at multiple sites: Thr181, Ser202/Thr205, Thr212, Thr231, Ser262, Ser396/Ser404, Ser409 and Ser422. Most of these sites were phosphorylated to a lesser extent in S2 than in P3 fractions. Tau polymers were detected in P3 fractions from 3-month and older female JNPL3 mice, but not in non-transgenic controls. The results suggest that tau in S2 represents an intermediate from which insoluble tau is derived, and that phosphorylation may play a role in filament formation and/or stabilization.Journal of Neurochemistry 01/2003; 83(6):1498-508. · 4.06 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The paired helical filament, which comprises the major fibrous element of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated microtubule-associated protein tau. Many of the hyperphosphorylated sites in tau are serine/threonine-prolines. Here we show that the stress-activated protein (SAP) kinases SAPK1gamma (also called JNK1), SAPK2a (also called p38, RK, CSBPs, Mpk2 and Mxi2), SAPK2b (also called p38beta), SAPK3 (also called ERK6 and p38gamma) and SAPK4 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Based on initial rates of phosphorylation, tau was found to be a good substrate for SAPK4 and SAPK3, a reasonable substrate for SAPK2b and a relatively poor substrate for SAPK2a and SAPK1gamma. Phosphorylation of tau by SAPK3 and SAPK4 resulted in a marked reduction in its ability to promote microtubule assembly. These findings double the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.FEBS Letters 07/1997; 409(1):57-62. · 3.54 Impact Factor
An inhibitor of tau hyperphosphorylation prevents
severe motor impairments in tau transgenic mice
Sylvie Le Corre*†, Hans W. Klafki*‡, Nikolaus Plesnila§, Gabriele Hu ¨binger*, Axel Obermeier*, Heidi Sahagu ´n*,
Barbara Monse*, Pierfausto Seneci*¶, Jada Lewis?, Jason Eriksen?, Cynthia Zehr?, Mei Yue?, Eileen McGowan?,
Dennis W. Dickson?, Michael Hutton?**, and Hanno M. Roder*††
*Sirenade Pharmaceuticals, Am Klopferspitz 19a, 82152 Martinsried, Germany;§Experimental Neurosurgery Institute for Surgical Research,
Ludwig–Maximilian University Munich, Marchioninistrasse 15, 81377 Munich, Germany; and?Department of Neuroscience, Mayo Clinic,
4500 San Pablo Road, Birdsall Building 210, Jacksonville, FL 32224
Communicated by Vernon Martin Ingram, Massachusetts Institute of Technology, Cambridge, MA, April 14, 2006 (received for review September 22, 2005)
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.
Alzheimer’s disease ? extracellular signal-regulated kinase inhibitor ?
paired helical filament ? tangles
represent characteristic hallmarks of several neurodegenerative
disorders including Alzheimer’s disease (AD), frontotemporal de-
mentia with parkinsonism linked to chromosome 17 (FTDP-17),
and others (reviewed in ref. 1). Compelling evidence that tau
malfunction or dysregulation alone can be sufficient to cause
neurodegeneration comes from the identification of mutations in
the tau gene that cause FTDP-17 (2, 3).
an early and pivotal event in the pathogenesis of AD and other
tauopathies (4). Hyperphosphorylation interferes with the normal
function of tau by abrogating the ability of tau to stabilize and
promote the assembly of microtubules (5). Phosphorylation is
sufficient to induce this loss of function as dephosphorylation of
abnormal hyperphosphorylation of tau may play a role in the
pathogenesis of tauopathies by inducing microtubule network
breakdown, followed by neuritic atrophy and neurodegeneration.
Additionally, hyperphosphorylated and?or aggregated species of
tau may exert direct toxic effects on neurons (7). It is important to
recognize, however, that most phosphorylation-related antigenic
markers of tau also are found in the normal brain, if postmortem
in vitro by a broad range of proline-directed kinases (8). Indeed,
basal phosphorylation levels at some of these sites within tau are
thought to play an important role in the normal regulation of tau
function and microtubule dynamics (9).
Although it is clear that aberrant phosphorylation of the tau
protein is a defining and invariable feature of the neurofibrillary
phosphorylation patterns has hindered the unambiguous identifi-
cation of the relevant protein kinases contributing to both normal
ious kinases have been implicated specifically in abnormal hyper-
eurofibrillary lesions composed of hyperphosphorylated and
aggregated forms of the microtubule-associated protein tau
phosphorylation of the 17 Ser?Thr-Pro motifs mainly responsible
for the diagnostic antigenic properties of pathological tau species
(reviewed in refs. 8 and 10). extracellular signal-regulated kinase
(ERK) 2 has been a particularly conspicuous candidate because of
its ability to phosphorylate all these sites up to the maximal
stoichiometry (11, 12), an ability matched only by SAPK4 and to a
slightly lesser degree by SAPK3 (ERK6) (13). Consequently even
disease-specific tau phosphoepitopes, such as those related to
certain Ser-422 phosphorylation-dependent epitopes (14), are fully
reconstituted in vitro by ERK2, but not by other known tau-kinases
like cdk5 or GSK3, which phosphorylate tau only to lower stoichi-
(15, 16) also is compatible with a role for ERKs in the pathological
hyperphosphorylation of tau.
Here we report a significant reduction in the levels of abnormal
hyperphosphorylated tau species and prevention of the severe
motor impairments in JNPL3 transgenic mice expressing P301L
mutant human tau after chronic treatment with a previously
selectivity. Our findings provide clear in vivo evidence that inhibi-
tion of pathological tau hyperphosphorylation can delay or prevent
as an approach to developing a disease modifying treatment for
tau-related neurodegenerative disease.
Despite extensive screening efforts for small molecule inhibitors of
ERK2, only the known natural product K252a produced useful
inhibitory activity for ERK2, a hitherto unappreciated property of
this otherwise well known compound (Fig. 1B). K252a was not
specific for ERK2 but had comparable activity against cdk1 and
moderate specificity (vs. PKC, PKA, and GSK3?) in vitro (G.H., S.
Geis, S.L.C., S. Gordon, R. Francasso, S. Ferrand, H.W.K., and
H.M.R., unpublished data). A medicinal chemistry program there-
fore was initiated to optimize the K252a lead structure for a proof
of concept experiment in tau transgenic mouse models (17, 18).
Conflict of interest statement: H.M.R. is a major shareholder of Tautatis, Inc., a company
developing inhibitors of tau pathology.
Abbreviations: AD, Alzheimer’s disease; ERK, extracellular signal-regulated kinase; NFT,
neurofibrillary tangle; NT, neuropil thread.
†Present address: AMO Germany GmbH, Rudolf-Plank-Strasse 31, 76275 Ettlingen,
‡Present address: Department of Psychiatry and Psychotherapy, University of Erlangen-
Nuremberg, Schwabachanlage 6, 91052 Erlangen, Germany.
Venzian 21, 20100 Milan, Italy.
**To whom correspondence should be addressed. E-mail: email@example.com.
††Present address: Tautatis, Inc., 124 Mount Auburn Street, Suite 200N, Cambridge,
© 2006 by The National Academy of Sciences of the USA
June 20, 2006 ?
vol. 103 ?
no. 25 ?
In keeping with the unusually restrictive inhibitor-binding site of
ERK2, apparent from the initial round of compound screening,
major structural alterations to the K252a scaffold did not yield
improved inhibitory activity. However, of all of the K252a deriva-
tives tested, those that inhibited ERK2 consistently blocked tau
hyperphosphorylation induced by okadaic acid in cell and brain
slice culture models (H. Roder, unpublished observations). SRN-
003-556 (Fig. 1A) is a synthetic indolocarbazole analogue of K252a
(Fig. 1B), which was substantially superior to K252a and other
analogs with respect to oral bioavailability and brain penetration.
However, the moderate selectivity of K252a (ERK2 ? cdk1 ?
PKA, GSK3, PKC) (G.H., S. Geis, S.L.C., S. Gordon, R. Fracasso,
S. Ferrand, H.W.K., and H.M.R., unpublished data) was essentially
lost with SRN-003-556. The compound inhibited ERK2, cdc2,
GSK3-?, PKA. and PKC in vitro in the presence of 250 ?M of ATP
with IC50values of 0.6, 0.18, 0.35, 0.44, and 0.26 ?M, respectively.
Pathological hyperphosphorylation of tau at multiple proline
directed serine and threonine sites, as induced by okadaic acid,
was potently prevented by SRN-003-556 in adult rat hippocampal
in inhibition of tau phosphorylation at the promiscuous AT8
phosphoepitope (site related to pS202?T205) and the patho-
specific AP422 phosphoepitope (site related to pS422) (mean
IC50 ? SD: AT8, 0.6 ? 0.19 ?M; AP422, 0.8 ? 0.18 ?M).
Additionally, the IC50 for a phosphoepitope related to the
nonproline-directed Ser-262 site in the microtubule-binding do-
main and not subject to direct ERK2 phosphorylation was nearly
coincident (anti-pS262: 0.5 ? 0.22 ?M). Despite the low kinase
specificity in vitro, 1 ?M SRN-003-556 did not induce metabolic
toxicity in SHSY5Y cells based on analysis of intracellular ATP
concentrations, whereas an in vitro cell viability assay with primary
human hepatocytes indicated cytotoxicity with an EC50of 22 ?M.
To adjust the oral dosing of the compound to match the target
concentration in brain as determined by the brain slice model
critical pharmacokinetic parameters relating to half-life in plasma
and Cmaxin plasma and brain were determined in C57BL?6NCrl
mice (Charles River Laboratories). Maximal concentrations of 1.8
?M in the brain, 5 ?M in the spinal cord, and 3 ?M in the plasma
(n ? 3) were detected 1 h after oral dosing of 30 mg?kg SRN-003-
556 in food-deprived mice. Compound levels declined at 3 h after
in the plasma. Compound levels in all tissues dropped to ?0.5 ?M
after 6–8 h. Oral bioavailability (26%) and good brain penetration
oral gavage at a dose of 15 mg?kg were 1.65 ?M and 2.6 h,
of tauopathy in vivo, we used the well characterized JNPL3 trans-
genic mice expressing mutant human P301L 4R0N tau. Expression
of this FTDP-17-linked mutant form of tau in these mice results in
age-dependent development of NFTs, neuronal loss, and progres-
sive motor deficits (19). Importantly, the major hyperphosphory-
lated detergent-insoluble tau species, which accumulates in this
model, comigrates at an apparent molecular mass of 64 kDa with
hyperphosphorylated forms of the 4R0N splice-isoform in deter-
gent-insoluble tau preparations from human AD brains (so called
‘‘PHF’’ tau) on SDS?PAGE, and displays the typical phospho-
epitope profile of PHF-tau (20). These observations suggest that
the biochemical mechanism of tau hyperphosphorylation in AD is
recapitulated in an authentic way in the JNPL3 mice.
For the dosing study, 72 hemizygous female transgenic JNPL3
mice were divided into two matched groups. Treatment with
one mouse from each cohort showed initial signs of motor deficits
study design was based on the remaining 70 JNPL3 mice from the
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 ?l
of SRN-003-556 (3.2 mg?ml in PEG 400; 10 mg?kg) at 1000 hours and another
200 ?l (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
was highly significant (P ? 0.0033; log-rank test).
SRN-003-556 delays?prevents the onset of motor deficits in JNPL3
SRN-003-556 (A) is an orally bioavailable CNS-penetrating synthetic analog of
Chemical structure and pharmacokinetics of SRN-003-556. (A and B)
tion in rat hippocampal slices in a dose-dependent manner. Four hundred
artificial cerebrospinal fluid buffer. SRN-003-556 was added 90 min before
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.
SRN-003-556 prevents okadaic acid-induced tau hyperphosphoryla-
www.pnas.org?cgi?doi?10.1073?pnas.0602913103Le Corre et al.
compound and vehicle-treated cohorts (n ? 35) developing ‘‘se-
vere’’ motor deficits as a phenotypic endpoint. ‘‘Severe’’ motor
impairment was defined by a dual set of tests, i.e., a wire hang test
and a beam balance test (see Materials and Methods for experi-
mental details). Composite failure criteria were chosen that were
sufficiently stringent to exclude false positives (i.e., mice that
recover after incidental performance slips). Under these criteria,
during a 9-week treatment period, with a twice daily oral dosing
regimen of alternating 10 and 20 mg?kg doses, 10 of 32 mice from
the vehicle-treated control group developed severe motor impair-
ments with an onset of disease at 260.1 ? 14.6 days (mean ? SD).
In contrast, only one of 31 mice treated with SRN-003-556 reached
this severe phenotypic stage at the age of 275 days, indicating that
SRN-003-556 significantly delayed or prevented the development
of motor deficits (P ? 0.0033 log-rank test) (Fig. 3). At the
termination of the study, 12 mice from the control group and 20
from the treated group did not show any signs of motor impair-
a mild or intermediate phenotypic stage after visual independent
inspection by two investigators. Mild or intermediate stages were
classified as motor deficits (e.g., abnormal gait or hind-limb scis-
soring) that did not reach the formal threshold used to define the
severe stage (see Materials and Methods for experimental details).
Blood and organs (liver, kidney, lung, stomach, and gut) of 20
animals across the phenotypic stages (n ? 10 per group) were
assessed for signs of toxicity, with special attention to potential
of organ and tissue pathology and no abnormal clinical chemistry
parameters, including pH, ions, hematocrit, and blood cell counts
(RBC, WBC, neutrophils, and platelets) were observed after
chronic 9-week exposure.
To gain insights into the mechanism of the prevention or delay
of the phenotype in the SRN-003-556-treated mice, we performed
neuropathological and biochemical analyses on brain and spinal
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
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).
Le Corre et al.
June 20, 2006 ?
vol. 103 ?
no. 25 ?
cord tissue from SRN-003-556-treated and control mice. To exam-
ine the phosphorylation state of tau proteins, spinal cord tissue was
homogenized in detergent-free kinase?phosphatase inhibitory
buffers followed by a low-speed centrifugation (13,000 ? g). The
supernatants then were subjected to Western blot analysis by using
a phosphorylation state-independent antibody (HT7) specific for
human tau and three phosphoepitope antibodies: AP422, AT8,
anti-pS262, and anti-pThr231 that recognize epitopes related to
pS422, pS202?T205, pS262, and pT231, respectively. Western blot
HT7, demonstrated the clear resolution of the pathologically hy-
perphosphorylated human tau band (64-kDa species) from the
mixture of normal human tau proteins (55- to 60-kDa species) by
virtue of its greatly retarded gel mobility. Signals of all tau-related
bands were quantified by densitometry and corrected for exposure
variation by reference to an appropriate phospho-tau standard on
each blot (see Fig. 4). Signals of the 64-kDa tau species were
normalized further to total human tau levels (55–60 kDa plus 64
in transgenic tau expression levels. Both phosphoepitope-specific
antibodies AP422 and AT8 detected only a single hyperphospho-
rylated tau species corresponding to the pathological 64-kDa tau
species. Importantly, the 64-kDa tau signals in spinal cord showed
Levels of the 64-kDa pathological phospho-tau species were sig-
control mice (AP422: 53% reduction; P ? 0.0072; AT8: 52%
reduction; P ? 0.0215; Fig. 4 B and C), although the average
amounts of total expressed human tau (55–60 kDa plus 64 kDa)
were not significantly different between the two groups (total HT7
signal, treated group: 88% of control; P ? 0.639). A similar link
between the amount of the 64-kDa tau species and levels of
phospho-tau detected by the anti-pT231 antibody also was noted
(data not shown). The anti-pS262 pAb and the HT7 mAb detected
not only the 64-kDa hyperphosphorylated tau species but also
normal tau proteins in the basal phosphorylation state between 55
and 60 kDa (Fig. 4A). The normalized 64-kDa tau signal with both
HT7 and anti-pSer262 also was reduced in the treated group to
approximately half the intensity found in the control cohort (anti-
phosphoepitopes (Fig. 4 D and E). However, the phosphorylation
state at Ser-262 of the normal tau proteins (55–60 kDa) was not
significantly different in the treatment (SRN-003-556) group vs.
controls (anti-pS262: 116% of control; P ? 0.8).
Formation of the 64-kDa pathological tau species also was
reduced to a lesser and not quite significant degree in low speed
(13,000 ? g) supernatants from hindbrain samples from the mice
(AT8: 26.5%; P ? 0.08) and similarly in 64-kDa tau-enriched
The smaller reduction in 64-kDa tau in the hindbrain compared
with the spinal cord may reflect a dose–response type relationship
by virtue of the lower levels of SRN-003-556 in brain vs. spinal cord
(based on the pharmacokinetic data for SRN-003-556 in brain vs.
spinal cord described above). Importantly, the pathological 64-kDa
tau species almost was completely removed from the low speed
(13,000 ? g) supernatant fraction by centrifugation at 150,000 ? g
(Fig. 5A). This finding demonstrates that this hyperphosphorylated
low-speed supernatant species constitutes mainly aggregated tau.
To examine the effects of SRN-003-556 on neuropathological
and semiquantitative analysis of NTs in SRN-003-556-treated and
vehicle-only mice. In the vehicle-treated cohort in this study, levels
of low-speed soluble 64-kDa tau species and NFTs and NTs in the
spinal cord increased in parallel with disease progression (Fig. 5B).
The largest increases in NFTs, NTs, and 64-kDa tau all occurred
with the phenotypic transition from the unaffected to the moder-
ately affected state. Accordingly, in the control mice, spinal cord
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
ical 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 pheno-
typic stage (0, unaffected; 1, mild-moderate; 2, severe).
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).
Chronic treatment of JNPL3Hlmc transgenic
www.pnas.org?cgi?doi?10.1073?pnas.0602913103Le Corre et al.
NFT numbers correlated with disease stage (r ? 0.72; Fig. 5D).
Surprisingly, a clear break in the relationship between the his-
topathological and the biochemical markers becomes apparent in
the comparison of spinal cord NFT counts in treated vs. vehicle-
only JNPL3 mice where treatment with SRN-003-556 did not
appear to reduce tangle numbers (Fig. 5C vs. Fig. 4 B–E) despite
the observed reduction in the aggregated 64-kDa pathological tau
species on Western blots described above. There was a nonsignif-
icant trend toward higher NFT counts in the treated group as a
whole (Fig. 5C) and, indeed, when sorted according to phenotypic
stage, it became apparent that the unaffected and moderately
affected mice treated with SRN-003-556 had a 2.9-fold and a
1.7-fold higher NFT count than the respective control groups,
although this increase again did not reach significance (unaffected,
P ? 0.071; moderately affected, P ? 0.185; Fig. 5 D and E).
Consequently, the moderately affected mice in the SRN-003-556-
treated group had NFT counts similar to those of severely diseased
control mice. In contrast, the levels of the 64-kDa tau species,
measured by Western blotting, were not significantly different
between treated and control mice within phenotypically matched
groups (data not shown) and correlated with phenotypic stage in
both the treated and control mice.
The data set of this study is consistent with the long suspected role
of abnormal hyperphosphorylation in the formation and?or stabi-
biochemical criteria) and their role in the onset of functional
After 9 weeks of treatment with the kinase inhibitor SRN-003-
556, we observed a significant delay or prevention of the typical
motor impairments in JNPL3-transgenic mice and a concomitant
selective reduction in pathological 64-kDa tau species, with its
complement of pathological phosphoepitopes. This pathological
64-kDa form of tau represents a distinct low-speed soluble but
high-speed sedimentable pool of aggregated tau. Given that trans-
genic mutant tau is the sole disease causing entity in this model, it
is reasonable to assume that the prevention of the onset?
progression of motor deficits is related causally to this inhibition of
abnormal tau hyperphosphorylation. However, in view of the
limited selectivity of the SRN-003-556 kinase inhibitor, we cannot
exclude that the therapeutic effects of the compound on the onset
of motor deficits in JNPL3 mice are modified by unrelated phar-
The observation that neither the average amounts of total
expressed human tau (55–60 ? 64 kDa) nor the phosphorylation
state at Ser-262 of the normal tau proteins (55–60 kDa) were
significantly different in the treatment (SRN-003-556) group vs.
controls is evidence for a degree of specificity for the inhibition of
tau hyperphosphorylation by SRN-003-556 in vivo, namely a dif-
ferential effect of the kinase inhibitor, despite its limited selectivity,
on the formation of the anti-pSer262 reactive pathological 64-kDa
tau species, but there was no effect on the basal phosphorylation
state at Ser-262 in normal tau proteins (55–60 kDa). This differ-
entiation of abnormal from normal tau phosphorylation events in
vivo is important in view of the likely role of phosphorylation in
regulating the normal function of tau.
Based on numerous pathological studies, it is generally assumed
that (i) abnormal hyperphosphorylation of tau is more or less
directly related to its aggregation, (ii) the biochemically defined
sarkosyl-insoluble pathological tau pool reflects the histopatholog-
ically defined NFTs and NTs, and (iii) the NFTs and NTs are direct
observations reported here indicate that the link between the
formation of such biochemically defined abnormal tau species and
histologically defined NFTs may be less direct than previously
assumed. The functional benefits we observed in JNPL3 mice after
9 weeks treatment with SRN-003-556 track with the reduction in
counts, which apparently continued to develop despite partial
inhibition of pathological tau hyperphosphorylation. It appears
likely that the roughly 50% inhibition of the formation of the
hyperphosphorylated 64-kDa tau species is sufficient to reduce?
prevent the neuronal dysfunction and neurodegeneration that
underlies the onset of the motor phenotype in JNPL3 mice but is
insufficient to retard the formation of NFTs or to reduce the
number of neurons developing tangles. This scenario could reflect
the amyloid-like nature of tau filaments that likely require lower
levels of pathological tau proteins to drive continued aggregation
after initial nucleation; however, we also cannot exclude the pos-
sibility that less pathological tau is incorporated into the same
number of developing NFTs in SRN-003-556-treated mice.
The results in the JNPL3 mice treated with SRN-003-556 are
consistent with the continued NFT formation observed in trans-
genic mice after suppression of 90% of mutant tau expression,
despite remarkable functional improvement and inhibition of neu-
ronal cell death (21). In addition, it has recently been observed in
the Htau transgenic mouse model of tauopathy that neuronal cell
death occurs independent of histologically observed tangles (22).
These previous studies and our own data suggest that NFTs are not
the main toxic entity in tauopathy, a situation reflected by recent
insights in the area of A? peptide toxicity (23) and other CNS
protein aggregation diseases, like CJD and Huntington’s disease
logical lesions are not the major cause of neurodegeneration and
the development of clinical symptoms. At this point, the relation-
ship between the biochemical and histological features of tau
pathology may be understood best in terms of different pools of
hyperphosphorylated tau. The relatively nontoxic tangle pool
seems to represent the stable default fate but has a limited rate or
accumulates in a lower molecular mass aggregated form and is
likely the major neurotoxic entity. These pools may exist in a
product?precursor relationship but also may constitute alternative
fates of hyperphosphorylated tau species. The exact role of hyper-
phosphorylation in the formation of various tau polymers remains
controversial, and our data does not directly address whether
hyperphosphorylation is required for initial aggregation or is a
secondary event perhaps involved in the stabilization of aggregated
tau. In any case, the ability of the kinase inhibitor SRN-003-556 to
delay or prevent the onset of severe motor deficits in JNPL3 mice,
without obvious harmful side effects, would predict a favorable
scenario for therapeutic intervention in human tauopathy with
kinase inhibitors, even if of limited specificity.
Materials and Methods
Preparation and Treatment of Rat Hippocampal Slices. Four hundred
fifty-micrometer hippocampal slices were prepared from 250- to
300-g male Wistar rats and preincubated for 20–30 min in oxygen-
ated ice cold, artificial cerebrospinal fluid with low Ca2?(26 mM
NaHCO3?3.5 mM KCl?1.5 mM KH2PO4?1.4 mM MgSO4?0.01
another 35 min at 1.3 mM Ca2?. SRN-003-556 was added at
Slices were extracted with homogenization buffer (500 mM Hepes,
pH 7.0?100 mM sodium pyrophosphate?2 mM EGTA?2 mM
EDTA?2 mM sodium orthovanadate?1 mM DTT?1 ?M okadaic
by sonication (10–15 s) followed by centrifugation (13,000 ? g at
4°C for 30 min).
SRN-003-556 Pharmacokinetics. Three to four female C57BL?6NCrl
mice (Charles River Laboratory; 20–25 g) were randomly assigned
per time point (0.5, 1, 3, 6, 8, and 24 h). Animals were deprived of
food intake 4 hours before oral administration of SRN-003-556 of
Le Corre et al.
June 20, 2006 ?
vol. 103 ?
no. 25 ?
and spinal cords. SRN-003-556 was extracted from 100-?l samples
200 ?l of saturated NaCl solution. The mixture was extracted twice
with 2 ml of ethylacetate (HPLC grade).
SRN-003-556 was extracted from CNS tissue by homogenization
?l concentrated of ammonia, extraction then was performed as
with the plasma samples. Extraction efficiencies (usually 90–95%)
were determined with plasma and CNS tissue homogenates spiked
with known amounts of SRN-003-556. Quantitative analysis of the
organic extracts was performed by HPLC with fluorescence detec-
tion at 284 nm (excitation) and 476 nm (emission) on a 5-?m RP
18 Select B 12.5 ? 4.6 mm column (Merck) at a flow rate of 0.75
ml?min with isocratic elution at a temperature of 40°C with
acetonitrile?water 60?40 (vol?vol).
Treatment of JNPL3 Mice with SRN-003-556.Seventy-twoheterozygous
into two matching groups. Treatment with SRN-003-556 or vehicle
(PEG 400) started at the age of 229 days. The compound was given
by oral gavage twice a day to food-deprived mice at 10 mg?kg (100
?l of solution in PEG 400) and 20 mg?kg (200 ?l of solution),
respectively. During the course of the study, seven mice died
without evidence of motor dysfunction (n ? 3 in control group and
Motor function was monitored twice a week by wire hang, beam
walk, and flight reflex tests and then once a day when the first signs
of motor impairments became evident. Animals who showed at
least two consecutive hang-test failures for two consecutive days
and?or drops from the beam two consecutive times for two
consecutive days and?or the animal failed once at the hang test and
simultaneously falls once from the beam for two consecutive days
were rated as having severe motor deficits and were killed. The
remaining mice that appeared either unaffected throughout the
whole study period or developed mild motor abnormalities below
formal threshold criteria for removal were killed at the end of the
treatment effect between the groups was assessed by a one-way
ANOVA Fisher’s post hoc test.
Mice were deeply anesthetized with 150 mg?kg of a Ketamine?
Xylazine mixture i.p. (Amersham Pharmacia, Upjohn, and Bayer),
and brains were resected quickly after removal of the skullcap;
spinal cord tissue as well as blood and other organs were collected
thereafter. One brain hemisphere was fixed in 4% paraformalde-
hyde, and the other half was immediately homogenized by sonica-
tion in ice-cold homogenization buffer for Western blot analysis.
Western Blot Analysis of Brain Slice, Brain, and Spinal Cord Superna-
tants. The proximal part of resected spinal cords (?2 cm) was fixed
in 4% paraformaldehyde for neuropathological studies, whereas
the remaining (distal) part and the hindbrains were subjected to
Western blot analysis. CNS tissues were extracted with 300 ?l of
by centrifugation (13,000 ? g at 4°C for 15–30 min). Supernatants
were adjusted to equal protein concentration, resolved by SDS?
PAGE on precast 10% Tris?Glycine gels (Anamed, Darmstadt,
Germany) and transferred to nitrocellulose membranes (Amer-
at room temperature with either 5% skim milk?3% BSA?TBST
(1? TBS plus 0.05% Tween 20) or 3% BSA?TBST depending on
the antibodies used. After incubation with primary antibodies
under blocking conditions, proteins were detected with secondary
antibody (peroxidase-linked anti-rabbit or anti-mouse IgG) and
enhanced chemiluminescence (ECL; Amersham Pharmacia Bio-
tech). For secondary probing of blots, previously bound antibodies
were removed in stripping buffer (0.2 M glycine, pH 2.5?0.05%
Tween) for 30 min at 60°C, and membranes were thoroughly
washed in TBST. Tau immunoreactive bands were quantitated by
digital densitometric scanning (Bio-Rad QUANTITY ONE imaging
software). Tau phosphoepitope signals on separate blots were
corrected for exposure by referencing to an appropriate phospho-
tau standard on the same blot: recombinant htau40, exhaustively
phosphorylated in vitro by a mixture of ERK2 and PKA, for AP422
(gift of M. Hasegawa, Tokyo Institute of Psychiatry, Tokyo) and
AT8 (Innogenetics, Gent, Belgium); for anti-pSer262 (BioSource
International, Camarillo, CA), an amount of 2 ?g of total protein
lysate of rat hippocampal slices treated with 1 ?M okadaic acid
(Sigma-Aldrich) as above. Signals were normalized further to total
human tau expression and loading by probing stripped blots either
with HT7 (Innogenetics) or with Tau-1 (Chemicon) after exhaus-
tive dephosphorylation on the blot by incubation in 50 mM
Tris?HCl, pH 8.5?0.1 mM EDTA?0.5 mM MgCl2containing 100
units?ml calf intestinal alkaline phosphatase (Promega) for 2 h
Extraction and Analysis of Sarkosyl-Insoluble Tau from Hindbrain.
Sarkosyl insoluble tau was prepared as described in ref. 19 and
analyzed by Western blotting as described above.
Neuropathological Analysis of Tau Lesions in Spinal Cord. Paraffin-
embedded tissue sections of spinal cord were stained with the
The number of neurofibrillary tangles in three nonoverlapping
microscopic fields from the anterior and intermediolateral gray
matter was recorded. Fields were chosen to reflect the highest
density of lesions. The density of argyrophilic neuropil threads was
2, moderate; 3, frequent.
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