Tau suppression in a neurodegenerative mouse model improves memory function

Abstract

Neurofibrillary tangles (NFTs) are the most common intraneuronal inclusion in the brains of patients with neurodegenerative diseases and have been implicated in mediating neuronal death and cognitive deficits. Here, we found that mice expressing a repressible human tau variant developed progressive age-related NFTs, neuronal loss, and behavioral impairments. After the suppression of transgenic tau, memory function recovered, and neuron numbers stabilized, but to our surprise, NFTs continued to accumulate. Thus, NFTs are not sufficient to cause cognitive decline or neuronal death in this model of tauopathy.

Fig. 1

Molecular and neuropathological characterization of rTg(tau_P301L)4510 mice. (A) The location of tau_P301L mRNA was analyzed by in situ hybridization and found to be largely restricted to the hippocampus, cortex, olfactory bulb, and striatum. (B) The amount of tau_P301L protein in total forebrain homogenates of a 2.5-month-old rTg(tau_P301L)4510 mouse was ~13 times the endogenous tau in a littermate expressing the activator only, assessed by immunoblotting. (C) Tau pathology was evaluated by using a panel of antibodies, including tau conformation–specific MC1 and tau phosphoserine 409 PG5, and histological methods, including modified Bielschowsky and Nissl with periodic acid Schiff stains. These analyses revealed conformational changes, followed by nonargyrophilic, hyperphosphorylated pretangles and culminating in abundant argyrophilic tangles and neuronal loss (CA1 hippocampal subfield is between the arrows). (D) Tangle pathology in a 10-month-old mouse, examined by electron microscopy, consists of straight filaments, which sometimes formed a herringbone pattern (inset) identical to that previously reported in the JNPL3 mouse (). Scale bar, 2 μm. (E) Gross forebrain atrophy, with preservation of hindbrain structures, in a 10-month-old tau_P301L-positive transgenic mouse (left) compared with a nontransgenic littermate (right).

Fig. 2

Neurodegeneration and suppression of transgenic tau. (A) Brain weights of tau_P301L-positive mice <5 months were significantly lower than those of tau_P301L-negative littermates (P < 0.001) (upper). Brain weights were significantly lower in 5.5-month-old and ≥7-month-old tau_P301L-positive mice, relative to younger mice (lower). *P < 0.05; **P < 0.001. (B) CA1 neuron estimates in one hippocampal subfield (n = 3 per group) were significantly lower in tau_P301L-positive mice 5.5 months of age and older. [ANOVA for genotype, F(1,16) = 23.97, P < 0.001]. *P < 0.05, **P < 0.01. (C) Transgenic tau_P301L mRNA levels, whether normalized to GAPDH or to murine tau mRNA levels, declined with age and were dramatically suppressed after brief exposure (6 to 8 weeks) to doxycycline. (D) Sarkosyl-soluble tau_P301L levels, measured in extracts from forebrain homogenates by using immunoblots, declined with age and were sbstantially reduced in animals receiving doxycycline. (E) In situ hybridization: A (top left) 7-unit tau_P301L-expressing mouse; B (top right) 2-month-old 13-unit tau_P301L-expressing mouse; C (lower left) 4-month-old 13-unit tau_P301L-expressing mouse; D (lower right) 4-month-old 13-unit tau_P301L-expressing mouse treated with doxycycline for 6 weeks. In this and subsequent figures, data are presented as means and SEM, and dashed lines compare groups of animals at the beginning and the end of doxycycline administration.

Fig. 3

Neuron death ceases and memory recovers when transgenic tau expression is suppressed. (A) Brain weights in 9.5- to 10-month-old tau_P301L-positive mice exposed to doxycycline for 4.5 months were significantly higher than brain weights in 7-month-old tau_P301L-positive mice but were not higher than those of tau_P301L-positive mice at 5.5 months, the age at which doxycycline was initiated. *P < 0.01. (B) CA1 neuron numbers in one hemisphere were estimated and found to stabilize after doxycycline treatment. (C) Twenty mice with target quadrant occupancies <39.5%, making up the half of a 2.5-month-old cohort having lower performance ratings, were tested in the water maze a second time at 4.5 months in the presence (n = 10) or absence of doxycycline (n = 10), which was initiated at 2.5 months. In tau_P301L-positive mice, memory recovered significantly when transgenic tau was suppressed. In tau_P301L-negative mice with target quadrant occupancies in the half of a cohort at 2.5 months of age having lower performance ratings in the presence (n = 13) or absence (n = 31) of doxycycline, a trend toward improved performance at 4.5 months was due to regression toward the mean (~48% in tau_P301L-negative mice), as the trend was not apparent when the complete cohort was examined. *P < 0.05. (D) Memory was tested longitudinally at four ages in 15 mice in the top performing half of their cohort at 2.5 months of age (target quadrant occupancies >39.5%). Doxycycline was administered at 5.5 months to 10 mice, and control feed was administered to 5 mice. Significant improvements were found in tau_P301L-positive mice treated with doxycycline, but not in tau_P301L-positive mice maintained on the control diet. *P < 0.05, **P < 0.01, ***P < 0.0001.

Fig. 4

Accumulation of NFTs after transgenic tau suppression. (A) Semiquantitative grading of neurofibrillary pathology in tau_P301L-positive mice. Tangles in three to five animals, at ages in months (M), evaluated with PG5, AT8, or Bielschowsky stain, in neocortical (ctx) and CA1 hippocampal (CA1) regions were graded using an adaptation of the CERAD (Consortium to Establish a Registry for AD) neuropsychological rating scales, by using area of most severe involvement, as follows: 0 (none), + (sparse, <5 NFTs/hpf), ++ (moderate, 5 to 25 NFTs/hpf), or +++ (severe, >25 NFTs/hpf). The neuropil scored 0 when AT8 staining was absent, + when only a mild granular background staining was noted, ++ when neuropil staining was moderate, and +++ when neuropil staining was severe and diffuse. (B) CA1 neurons staining with PHF1 antibody recognizing tau phosphoserines 396 and 404 did not diminish after transgene suppression for 6 weeks. (C) Concentrations of sarkosyl-insoluble tau_P301L proteins were measured in extracts of forebrain homogenates by using immunoblots. With age, the predominant tau species shifted from a doublet of ~55-kD proteins to a 64-kD protein. Suppression of tau_P301L reduced levels of the 55-kD species (gray symbols), but failed to halt the accumulation of the 64-kD species in mice ≥4 months of age (black symbols). (D) The number and length of fibrils in sarkosyl-insoluble fractions from extracts of forebrain homogenates increased with age. Suppressing transgenic tau in ≥4-month-old mice failed to prevent the increase in the amount or size of the fibrils.