Passive immunization with phospho-tau antibodies reduces tau pathology and functional deficits in two distinct mouse tauopathy models.

In Alzheimer's disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies

Model of antibody engagement of tau in-vivo.

<p>A compartmental model depicting tau and antibody (IgG) levels in brain, interstitial fluid (ISF), cerebrospinal fluid (CSF) and plasma. CSF tau is truncated with levels of ~1 nM, while tau in ISF exists as a full-length molecule with levels of 3–5 nM (4-5x greater than in CSF) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125614#pone.0125614.ref047" target="_blank">47</a>]. Concentrations of p-tau are estimated to be about 1–10% of total tau levels. Tau antibody concentrations are 1–3 nM in CSF and ISF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125614#pone.0125614.ref044" target="_blank">44</a>]. Antibody engagement of p-tau in CSF and ISF would enable clearance of tau via a variety of antibody-mediated mechanisms. Full-length tau is indicated as a molecule containing N-terminal (orange line), mid-domain (blue line), microtubule-binding repeat region (black box), and C-terminal (grey line) regions, whereas truncated tau in the CSF compartment is indicated by the mid-domain and N-terminal fragments.</p

Age-dependent changes in tau pathology in rTg4510 mice.

<p><b>A.</b> AT8 p-tau staining in 3- and 6-month old rTg4510 brain sections. Scale bar = 500 μm. <b>B-E.</b> Age-dependent change in <b>B.</b> hippocampal soluble total tau, <b>C.</b> hippocampal soluble AT8 p-tau, <b>D.</b> hippocampal insoluble total tau, and <b>E.</b> hippocampal insoluble AT8 p-tau. Individual comparisons were vs. 3.5-month old mice using ANOVA with a Dunnett’s post-hoc test (*p<0.05, ** p<0.01, *** p<0.001; n = 7–11 per group). <b>F.</b> Hippocampal soluble AT8 levels normalized to total tau levels were correlated with AT8 staining scores from immunohistochemistry (R² = 0.51, p<0.001).</p

PHF13 reduces EC tau pathology and rescues NOR performance in PS19 mice injected with K18PL PFFs.

<p>K18PL PFFs were injected into the hippocampus and mice evaluated following treatment with PHF13 or IgG2b for 4 weeks. <b>A.</b> Hippocampal area occupied by AT8 immunostaining on the ipsilateral (Ipsi) and contralateral (Contra) sides following treatment with 30 mg/Kg i.p. IgG2b or PHF13. <b>B.</b> AT8-positive cell counts within the EC of IgG2b- and PHF13-treated mice. <b>C.</b> MC1-positive cell counts within the EC of IgG2b- and PHF13-treated mice. <b>D.</b> Percent time spent on the novel object in a NOR assay. Statistical analyses were performed using a t-test comparison between IgG2b and PHF13 treatment groups (* p<0.05, ** p<0.01; n = 10-12/group).</p

Correlations between brain and CSF tau and p-tau levels.

<p>Correlations between brain and CSF tau and p-tau levels.</p

p-Tau antibodies reduce brain p202/p205 tau and CSF p181 tau with improved NOR performance.

<p>Effect of PHF-13 and PHF-6 (25 mg/Kg i.p.) on <b>A.</b> hippocampal soluble total tau; <b>B.</b> hippocampal soluble AT8 (p202/p205) tau; <b>C.</b> hippocampal insoluble total tau; <b>D.</b> hippocampal insoluble AT8 tau; <b>E.</b> CSF total tau; and <b>F.</b> CSF pT181 tau. Data were analyzed by ANOVA with post-hoc comparisons to IgG2b-treated mice using Dunnett’s test (*p<0.05, ** p<0.01; n = 14-18/ group). Red dashed lines indicate tau levels at the start of treatment (3-months of age). <b>G.</b> Effect of p-tau antibodies on NOR performance. The total time spent on novel (filled bar) and familiar objects (open bar) are plotted for each treatment group. DN represents double-negative (-tTA and—tau) mice. Statistical comparisons between the time spent on novel and familiar objects for each animal in a group was performed using a pairwise T-test. (DN, p<0.05; IgG2b p = 0.8; PHF13, p<0.05; PHF6, p<0.01; n = 6-8/group).</p

PHF13 reduces contralateral hippocampal tau pathology in PS19 mice injected with K18PL PFFs.

<p>The extent of hippocampal MC1-positive tau pathology was evaluated in PS19 mice that received K18PL PFF injections into both the hippocampus and overlying cortex and treatment with either IgG2b or PHF13 (30 mpk i.p. for 4 weeks). <b>A.</b> Contralateral hippocampal images from coronal sections stained with MC1 from mice treated with either IgG2b (top panels) or PHF13 (bottom panels). Right panels show images after non-biased thresholding to identify MC1-positive pathology for quantification. <b>B.</b> The percent of hippocampal area occupied by MC1 staining from IgG2b- and PHF13-treated mice. The left Y-axis and panels (black symbols) show ipsilateral (Ipsi) hippocampal (HP) % area, whereas the right Y-axis and panels (red symbols) show contralateral (Contra) hippocampal % area. Statistical analyses were based on t-test comparisons between IgG2b and PHF13 treatment groups (** p<0.01).</p

Immunodepletion with p-Tau antibodies reduces tau pathology in a primary neuronal model of tau aggregation.

<p><b>A.</b> Kinetics of association and dissociation of PHF6 antibody and PHF13 antibody. The table summarizes the calculated association rates, dissociation rates, and K_D values for PHF6 and PHF13. The vertical dotted lines demarcate, from left to right—peptide loading, wash/equilibration, antibody association and antibody dissociation. <b>B.</b> Representative images from Triton-insoluble MC1 staining of primary neurons that received no PFF treatment (- PFF), treatment with 100 nM K18PL PFF (+ PFF), rTg4510 brain extract (Tg4510) or tTA brain extract (tTA). <b>C.</b> Change in Triton-insoluble MC1 staining intensity in primary neurons treated as in B, relative to untreated cultures (n = 6 wells/condition; ANOVA with Dunnett’s test vs. no treatment). <b>D.</b> Immunodepletion of rTg4510 extracts with PHF6 or PHF13 led to significant declines in Triton-insoluble MC1staining (Red bar) in neurons relative to control antibody (ANOVA with Dunnett’s test vs. Control IgG). Also displayed are total tau (black bar) and p-tau levels (blue bar) measured by ELISA after immunodepletion (*p<0.05, *** p<0.001, n = 3 per group).</p