In Vivo Tau Imaging for a Diagnostic Platform of Tauopathy Using the rTg4510 Mouse Line

Association of tau deposition with neurodegeneration in Alzheimer’s disease (AD) and related tau-positive neurological disorders collectively referred to as tauopathies indicates contribution of tau aggregates to neurotoxicity. The discovery of tau gene mutations in FTDP-17-tau kindreds has provided unequivocal evidence that tau abnormalities alone can induce neurodegenerative disorders. Therefore, visualization of tau accumulation would offer a reliable, objective index to aid in the diagnosis of tauopathy and to assess the disease progression. Positron emission tomography (PET) imaging of tau lesions is currently available using several tau PET ligands. Because most tau PET ligands have the property of an extrinsic fluorescent dye, these ligands are considered to be useful for both PET and fluorescence imaging. In addition, small-animal magnetic resonance imaging (MRI) is available for both structural and functional imaging. Using these advanced imaging techniques, in vivo studies on a mouse model of tauopathy will provide significant insight into the translational research of neurodegenerative diseases. In this review, we will discuss the utilities of PET, MRI, and fluorescence imaging for evaluating the disease progression of tauopathy.

inTRODUCTiOn Dementia is a leading cause of death in developed countries. Increasing age is the greatest risk factor for dementia. About 46 million people in the world are estimated to be suffering from dementia, and this figure is expected to rise to 135 million by 2050. Alzheimer's disease (AD) is the most common type of dementia. The global impact of AD will strikingly affect social and economic costs. Distinctive features of AD are the deposited accumulations of β-amyloid (Aβ) protein fragments and intracellular neurofibrillary tangles (NFTs) (1). NTFs are closely associated with the severity of brain function loss in AD (2). Therefore, making tau protein a target in the treatment of AD has become a major therapeutic strategy.
Recent advances in positron emission tomography (PET) imaging research have led to significant breakthroughs with the application of newly developed tracers for visualizing regional tau depositions (3)(4)(5)(6)(7)(8). This technology allows us to non-invasively evaluate the progression of tau pathology in living brains. PBB3 was developed as a novel PET tracer that binds with tau for the diagnosis of AD and other neurodegenerative diseases regarded as tauopathies (4, 9). PET imaging using [ 11  (radiotracer) is superior in detecting tau deposits, and it is closely aligned with disease symptoms. This was also confirmed by our tauopathy mouse models (4; Ishikawa, forthcoming 1 ). 11 C-labeled tracers, due to their short half-life, are generally unsuitable for clinical practice, but [ 11 C]PBB3 can be used to evaluate certain tau conditions including AD (4, 10). The fluorescent quality of PBB3 makes it especially useful for multimodal imaging, allowing us to assess its binding by microscopic observation of related cells and tissues. Animal models of tauopathy are essential for preclinical studies of AD and related dementias. The rTg4510 mouse line was developed to model aspects of tauopathy through forebrain expression of the P301L mutated human tau (11). Expression of human tau is controlled by the tetracycline transactivator transgene under the CaMKIIα promoter. This mouse line develops progressive intracellular tau aggregations in corticolimbic areas and forebrain atrophy (11). The age-dependent tau pathology of rTg4510 mice has been investigated in detail by immunohistochemical and biochemical examinations (12)(13)(14). These postmortem brain-based studies showed an extensive increase of pathological tau inclusions in the cerebral cortex and hippocampus between 4 and 6 months of age. Now, for both current and therapeutic strategies, rather than endpoint measurements, researchers are eagerly involved in the development of new in vivo protocols for tracking the progressive pathological status in living animals.
Taking advantage of the multimodality of the PBB3 ligand, in vivo monitoring of NFT formation is now available for possible in tauopathy mouse models, including the rTg4510 mouse. In addition, advanced brain magnetic resonance imaging (MRI) techniques have enabled us to visualize neuronal dysfunction and the structural changes related to neurodegenerative processes in living animals. Here, we will introduce in vivo multimodal imaging technologies, including PET, MRI, and fluorescence imaging to investigate the real-time events of taurelated neuropathology.

MRi STUDieS FOR A MOUSe MODeL OF TAUOPATHY
Magnetic resonance imaging-based volumetry is a valuable tool for assessing disease progression in humans (15)(16)(17)(18)(19). As translational research of neurodegenerative diseases, several groups have reported studies using volumetric MRI in mouse models (20)(21)(22)(23)(24)(25). Age-dependent volume reduction in both cerebral cortex and hippocampus of the rTg4510 mouse line was clearly demonstrated on MRI (24,25). The volume (size) of the cerebral cortex from 5 to 8-month-old rTg4510 mice was significantly less (by approximately 20%) than that of age-matched non-transgenic (non-tg) mice (24). Brain atrophy in rTg4510 mice as examined by MRI was in agreement with previous histopathology findings (13,26). A gender difference showing more severe phenotype in female rTg4510 mice compared with males (27) was confirmed by volumetric MRI study (24).
In addition to volumetric MRI, MR-based in vivo imaging techniques, including MR spectroscopy (MRS), manganeseenhanced MRI (MEMRI), arterial spin labeling, amide proton transfer imaging, and diffusion tensor imaging (DTI), have been tested to evaluate brain functions and microstructural changes in living rTg4510 mice (24,25,(28)(29)(30)(31)(32). MEMRI is a technique for measuring neuronal function, because the manganese ion (Mn 2+ ) accumulates in actively firing neurons through voltage-gated Ca 2+ channels and serves as a positive contrast agent in T1-weighted brain images (33,34). Using this technique, Perez et al. observed a reduction in both neuronal activity and volume in the hippocampus of 6-month-old rTg4510 mice (25). Fontaine et al. demonstrated neuronal dysfunction in the CA3 and CA1 regions of 3-month-old rTg4510 mice (32). Together, these studies illustrate the importance of the MEMRI method as a diagnostic research tool for the early detection of neuronal dysfunction in rTg4510 mice.
Diffusion tensor imaging is another MR-based imaging technique, which is applied to assess the integrity and organization of myelinated structures such as axons in white matter (WM) and also in gray matter regions (35). For this purpose, we used DTI to examine age-related alterations in WM diffusion anisotropy in rTg4510 and non-tg control mice (29). Our results indicated that 8-month-old rTg4510 mice show significant reductions in fractional anisotropy (FA) in WM structures compared with control non-tg and young (2.5-month-old) rTg4510 mice. The microstructural changes contributing to these age-and tauopathy-associated WM changes were supported by electron microscopic evidence of disorganized axonal processes and the presence of interprocess spaces, which could explain the reduced FA in aged rTg4510 mice (29). Reduced FA at a later age (over 7.5 months) was confirmed by another research group (30,31). Since, reduced FA values in WM were mostly observed at an age that included the presence of significant atrophy and tau pathology, this standard method may be useful for assessing the therapeutic efficacy of treatments that may be used to reduce or prevent neurodegenerative progression. On the other hand, examination with additional diffusion anisotropy indices such as the mode of anisotropy allowed us to detect early signs (at 2.5 months old) of WM disorganization (29). Although disorganization of myelin morphology at this age was not determined, WM degeneration may be one of the early signs of tauopathy. Nevertheless, reduced WM integrity associated with tau pathology was confirmed by in vivo DTI studies.

TAU PeT TRACeRS
Current tau PET tracers are mostly designed by β-sheet binding properties. In principle, filamentous tau aggregates (e.g., NFTs, neuropile threads, tufted astrocytes, astrocytic plaques, and coiled bodies) will be labeled by these tracers with distinct specificity and selectivity. Up to date, three types of radiotracers have been widely tested for the clinical assessment of patients with tauopathy [reviewed in Ref. (36)]. These tracers include the arcyquinoline derivative THK5351 (37), the pyrido-indole derivative AV-1451  (38,39), and the phenyl/pyridinyl-butadienyl-benzothiazole/ benzothiazolium derivative PBB3 (4). Screening of potential tracers was performed by in vitro binding assays in AD brain homogenates and autoradiographies of AD brain sections.  (36,42,43). Although binding properties of tau PET tracers have been vigorously investigated for the past decade, the specificity and selectivity of PET signals to tau pathology are still to be fully understood.

TAU PeT iMAGinG STUDieS OF rTg4510 MiCe
Micro-PET imaging of tau pathology in tg mouse models of tauopathy has contributed to the characterization of novel PET tracers. Using [ 18 F]THK523, micro-PET imaging in 6-monthold rTg4510 mice successfully showed significantly higher radiotracer retentions in brains compared with non-tg or PS1/ APP mice (44). [  reach a few 100 μm. The imaging depth reached has been up to ~1 mm depending on the properties of the tissue (47). Using this technology, AD mouse models have been investigated with regard to monitoring the time course of disease progression [reviewed in Ref. (48)]. Hyman's group first reported in vivo visualization of amyloid plaques in APP tg mouse lines (PDAPP mice and tg2576 mice) (49,50). Clearance of plaques by anti-Aβ antibody was monitored by two-photon microscopy after Thioflavin S injection into the brain. Fluorescence imaging using Pittsburgh Compound B (PiB), which is used for amyloid PET imaging, was also examined to visualize amyloid plaques in APP tg mouse lines (51). As a result, the multimodality of PiB ligand for both fluorescence and PET imaging was clearly demonstrated. Because the PBB3 ligand was derived from the same tracer family as the PiB ligand, a similar approach was used to visualize the tau pathology in PS19 tg mice (4). Moreover, the current chronic cranial window setting enables the use of long-period two-photon imaging for more than 2 months (52). As for our study, longitudinal monitoring of PBB3-positive neurons has been performed in living rTg4510 mice (Takuwa et al., manuscript in preparation). Our data showed that PBB3positive inclusions were visualized with two-photon imaging at an age of as early as 4 months, with fluorescence signals then reaching a plateau at 6 months. These data are in agreement brain atrophy disappeared in rTg4510 mice over 7 months old. Since, tau pathology is tightly linked to neuronal loss, the disappearance rate of [ 11 C]PBB3-positive neurons may increase at age over 7 months. It should be noted that microglial activation in rTg4510 brains examined using translocator protein (TSPO) (18-kDa TSPO)-PET imaging showed significant correlation with both [ 11 C]PBB3 level and brain atrophy (see text footnote 1). rTg4510 will be a useful model for investigating the mechanisms of tau-induced neuroinflammation. Nevertheless, in combination with tau PET imaging, the imaging of neuroinflammation will offer an additional diagnostic parameter for tauopathy.

IN VIVO FLUOReSCenCe iMAGinG FOR DeTeCTinG TAU PATHOLOGY
The invention of two-photon excitation laser scanning microscopy has given new impetus to the research field of investigating in vivo brain cell dynamics (46). In combination with newly developed fluorescence imaging techniques, cellular and molecular mechanisms underlying brain functions and impairments can be examined using in vivo animal models. In theory, the spatial scales of two-photon microscopy are from a micron to a millimeter. The side length of field-of-views can In Vivo Imaging on a Model of Tauopathy  (53). Their data indicate that NFT formation has a neuroprotective effect against caspase-mediated neuronal death. Synaptic dysfunction is another key event taking place during the pathogenesis of tauopathy. Previous reports showed decreased dendritic spine and synapse density in cortical slices prepared from 9 to 10-month-old rTg4510 mice (54,55). Most recently, Jackson et al. demonstrated the visualization of synapses in the somatosensory cortex of rTg4510 mice by twophoton microscopy after the injection of adeno-associated virus that drove neuronal expression of either GFP or GCaMP6m (56). For longitudinal imaging, GFP-expressing axonal and dendritic regions were imaged weekly to investigate the turnover of axonal terminal boutons and dendritic spines. For functional imaging, GCaMP6-expressing neurons were imaged in lightly anesthetized animals to measure neuronal activity in response to whisker stimulation. The authors observed mismatched abnormalities in pre-and post-synaptic turnover coinciding with disrupted neuronal activity at 5 months of age. Their data suggests that synaptic dysfunction precedes tangle-associated neurodegeneration. Although linkage between aggregated tau formation and synaptic dysfunction remains unclear, in vivo monitoring of tau pathology at cellular levels provides an advantage for dissecting the mechanisms of tau toxicity.

DeSiGn FOR A DiAGnOSTiC PLATFORM OF TAUOPATHY
The rTg4510 mouse is one of the widely used models of tauopathy. As described above, several groups have conducted in vivo imaging studies on rTg4510 mice (Table 1). Because the time course of tau pathology and forebrain atrophy have been well examined using postmortem materials, experimental designs for drug intervention can be easily designed (Figure 1). Since, this mouse model allows the tetracycline-repressible overexpression of human tau, doxycycline treatment for the suppression of tau expression will provide a positive control in an experimental design of the evaluation of therapeutic candidates. Previous study showed that suppression of tau from 5.5 months of age reversed memory deficits, while tangles persisted and continued to accumulate (11). Therefore, early intervention starting at 2 months of age could be more effective to prevent both NFT formation and brain atrophy ( Figure 1B). Although effects will be limited, late intervention would be worthwhile if aggravation of tauinduced brain atrophy can be slowed down ( Figure 1C). In our study, we developed a unique diagnostic platform of in vivo imaging of volumetric MRI, tau-PET, and TSPO-PET ( Figure 1D). Brains of living rTg4510 mice can be used to monitor their volume, pathological tau accumulation, and neuroinflammation. Moreover, live cell imaging with two-photon microscopy allows us to capture NFT formation and neuronal death in rTg4510 mice. Using these platforms, we expect to be able to validate several drug candidates in the foreseeable future.

COnCLUSiOn
For more effective therapies, the pre-clinical evaluation of drugs for tauopathy is needed. The use of animal models that recapitulate the critical features of the disease, such as NFTs, cognitive impairment, brain atrophy, and neuronal loss, is essential. The rTg4510 mouse model of tauopathy fulfills the required features, despite the fact that tau protein was expressed at a non-physiologically higher level over the total life span. In vivo, brain imaging offers reliable approaches to validating the pathological status and to determining the efficacy of drugs for exploring disease-modifying therapies.

AUTHOR COnTRibUTiOnS
Authors wrote and proofed the manuscript.